JP2012518994A - Identification method - Google Patents

Abstract

  The present invention relates to a novel chemical substance having broad spectrum chemokine inhibitor (BSCI) activity and a use as an anti-inflammatory agent, and a method for discovering a pharmaceutical composition. In one embodiment, the method for identifying a compound or substance having BSCI activity comprises the following steps: (a) GPCR neuroreceptor in which one or more candidate compounds or substances are known to be expressed on leukocytes Then (b) compounds or substances that show binding to the selected GPCR neuroreceptor are tested for BSCI activity in a functional assay; and (c) to the selected GPCR neuroreceptor Compounds or substances that show binding to are tested for classical agonist activity in a functional assay, wherein step (a) is performed first, but steps (b) and (c) are in any order Or at the same time.

Description

  The present invention relates to a novel chemical substance having broad spectrum chemokine inhibitor (BSCI) activity and a use as an anti-inflammatory agent, and a method for discovering a pharmaceutical composition.

  Chemokines are a group of approximately 50 structurally related, mostly small (8-12 kDa) proteins that play an important role in the control of the mammalian immune system. Chemokine ligands signal through approximately 20 closely related receptors, and they are members of the type 1 class of G protein-coupled receptors (GPCRs). Importantly, many of the ligands bind to two or more receptors and signal productively through them. Similarly, many receptors bind to more than one ligand.

  Various subsets of these chemokine receptors are expressed on specific types of leukocytes. As a result of this complex degeneracy, precise temporal and spatial control of leukocyte recruitment is possible. Chemokines play a central role as the “traffic cop” of the immune system, encoding various complex white blood cells from the bone marrow through the blood to secondary lymphoid tissues or sites of immune response. there is a possibility.

  For this reason, compounds that modulate chemokine signaling have long been useful in a very wide range of diseases with inflammatory components (eg, atherosclerosis, autoimmune diseases, allergies, eczema, psoriasis, or neurodegenerative disorders) It is considered a promising candidate for the development of novel anti-inflammatory agents. There is a great need for new drugs for these diseases: existing small molecule therapeutics are generally effective but have severe side effects (eg corticosteroids) or less effective but compared Safe (for example, an NSAID such as diclofenac). Biotherapeutic agents (eg, antibodies to TNF-α) are more specific but are expensive to manufacture and are not suitable for simple oral administration. Small molecule chemokine inhibitors may have a better balance of efficacy and safety than other groups of small molecule anti-inflammatory agents, compared to existing biological anti-inflammatory agents. Gives chemistry (including cost).

  Unfortunately, specific chemokine receptor antagonists are slow to achieve this possibility. First they are found using traditional high-throughput molecular screening methods (eg, receptor binding assays for substances that can block ligand: receptor interactions, and random libraries are screened). Is difficult. Similarity between chemokine receptors means that compounds often react with a subset of chemokine receptors (instead of having specificity for a single receptor), and worse, many early chemokine receptors Modulators bind to serotonin and related receptors of the catecholamine system and cause undesirable side effects on behavior, blood pressure, and other phenotypes.

  Second, the excellent specificity for a single receptor within a class (which is the greatest goal of traditional drug development philosophy) may not be particularly effective even if this is ultimately achieved. It became clear. There are many related ligands that bind to the same receptor, and several receptors that can transduce signals for many ligands. Substances that block a single receptor are usually due to the degeneracy of the chemokine system It has no general anti-inflammatory properties and is weak enough to disappoint the immune system. This can be compared to a musical amplifier: anti-inflammatory effects require “volume control” regulation, while specific chemokine receptor antagonists act on “graphic equalizers”. That is, such substances slightly modulate the type of immune response (perhaps alter the cellular composition of the infiltrate by the leukocyte subset present) but do not quantitatively reduce the degree of inflammation.

  As a result, there are no chemokine inhibitors approved as human therapeutics even in 2009, more than 20 years after the chemokine system was first described, and then emerged as a major immunomodulatory target (but BX471, a specific antagonist of the CCR1 chemokine receptor in rheumatoid arthritis, for example, with some indications that are late in clinical trials with specific indications and modulation of the type of inflammatory response has some effect (Horuk (2005) Mini Rev. Med. Chem. 5: 791-804)). Therefore, another approach is needed: compounds that simultaneously block many chemokine signals (we broad-spectrum chemokine inhibitors, or substances named BSCI; Grainger & Reckless (2003) Biochem. Pharmacol. 65: 1027-34; Grainger et al. (2005) Mini Rev. Med. Chem. 5: 825-32).

  Compounds with broad spectrum chemokine inhibitor (BSCI) activity are a new class of agents that are useful as anti-inflammatory drugs for treating or preventing a wide range of diseases. In animal models of human disease, BSCI is associated with leukocyte accumulation and disease severity in atherosclerosis, stroke, asthma, surgical adhesion formation, endometriosis, dermatitis, endotoxemia, and rheumatoid arthritis. Proven to reduce other markers (Reckless et al. (2001) Immunology 103: 244-54; Beech et al. (2001) J. Cereb. Blood Flow Metabol. 21: 683 -9; Grainger & Reckless (2003) Biochem. Pharmacol. 65: 1027-34; Naidu et al. (2003) Ann. Thorac. Surg. 78: 1118-22; Naidu et al. (2004) J. Heart Lung Transplant 23: 128-34; Reckless et al. (2005) J. Vasc. Res. 42: 492-502; Berkkanoglu et al. (2005) Hum. Reprod. 20: 3047-52; Kayisli et al. (2007) Reprod. Sci. 14: 825-35; Hildebrandt et al. (2009), in print). BSCI has also been shown to suppress HIV replication in vitro (an anti-inflammatory activity independent of chemokine dependence of HIV cell entry; Grainger & Lever (2005) Retrovirology 2:23).

  Despite having potent anti-inflammatory activity in such a wide range of disease models, BSCI as a class is notably toxic or has low side effects (Schroff et al. (2005) Mini Rev Med Chem. 5: 849-55). For example, aminolactam BSCI has been evaluated by many routes of administration in a wide range of acute and chronic toxicity studies, but the level of inactivity is at least 1,000 times higher than the maximum therapeutic dose. These aminolactam BSCIs also show an excellent safety pharmacological profile (no effect on hERG tail current or no genotoxicity assay; see eg WO2009 / 016390).

  Taken together, these findings suggest that BSCI can provide an excellent tradeoff between efficacy and side effects compared to existing anti-inflammatory drugs (including corticosteroids and NSAIDs) as a class. Accordingly, there is a need for methods for identifying compositions having BSCI activity.

  Unfortunately, the BSCIs described to date have been fortunately identified by chance and there is no easy way to identify new, potentially structurally unrelated BSCIs. The first compound with BSCI activity is a peptide called Peptide 3, which was selected from the sequence of MCP-1 and tested for its ability to bind to multiple chemokine receptors (Reckless & Grainger ( 1999) Biochem. J. 340: 803-11). Peptide 3 was found not to bind to multiple chemokine receptors (but in fact binds to a single unidentified high affinity site on the cell surface; Grainger & Reckless (2003) Biochem. Pharmacol. 65: 1027-34) had BSCI activity against at least 5 chemokines among them capable of binding to at least 10 different receptors.

  From this peptide 3 structural template, a series of different structural groups of BSCI (including peptides, peptoids, and non-peptide structures) were designed, synthesized and characterized. Importantly, the structural motif responsible for the BSCI activity of the peptide is narrowed down to the WxQ tripeptide, where W is the amino acid tryptophan, x is any proteogenic amino acid, and Q is glutamine. That is. This allows the design of non-peptide WxQ mimetics, one of which (N-acyl glutarimide NR58,4) has nanomolar activity as BSCI (Fox et al. (2002) J. Med. Chem. 45: 360-70).

  NR58,4 is active in vivo as BSCI (having a strong anti-inflammatory effect in models of acute inflammation such as endotoxemia), but is not an ideal compound for drug development It was. Its essential imide ring is susceptible to hydrolysis by unspecified amidases in plasma, resulting in rapid inactivation of the compound in vivo (Fox et al. (2005) J. Med. Chem. 48: 867 -74).

  Later, further medicinal chemistry studies identified a related class of substituted aminolactams that retain the potency of BSCI but are stable in vivo. An example of this class of BSCI is N-oleoyl-3-aminocaprolactam (BN83253; Fox et al. (2005) J. Med. Chem. 48: 867-74). A wide range of other structurally related aminolactam BSCIs have also been described (eg, WO2005 / 053702, WO2006 / 016152, WO2006 / 018609, WO2006 / 085096; WO2006 / 134385, WO2006 / 134384, and WO2009 / 016390). However, at least some of them have properties suitable for development as pharmaceuticals for humans.

  Another group of non-peptide BSCIs has also been identified based on the structure of the essential WxQ pharmacophore. The alkaloid yohimbine (an alpha2-adrenergic receptor gonist obtained from the roots of Rauwolfia Serpentina) has a carbon skeleton similar to the specific conformation of the WVQ tripeptide, -Amide derivatives are highly active like BSCI (WO 99/12968; Grainger et al. (2005) Mini Rev Med Chem. 5: 825-32).

  A third group of non-peptide BSCIs has also been described, again based on the resergamide nucleus similarity to the carbon backbone of the WVQ tripeptide. Already reported risergamides, such as LSD (N, N-diethylamide of lysergic acid), which is a class A prohibited substance, act as agonists in multiple GPCRs (and thus have a wide range of physiological effects including hallucinogenicity). However, we have compound L-II (N-asparaginamide of dihydroresergic acid) and other glutamine and asparagine amide of lysergic acid as highly active as BSCI without the expected side effects of other lysergic acid amides. (WO99 / 12968).

  The structural relationships between all groups of BSCIs described to date are illustrated in FIG. From this figure it is clear that all groups of BSCI are identified by their similarity in chemical structure.

  There is currently no general method for identifying BSCIs that are not structurally related to known compounds. The reason for this limitation is the lack of information about the molecular target of BSCI. These BSCIs do not act as chemokine receptor antagonists and do not bind to any of the chemokine receptors tested, but instead bind to distinct (but not identified) high affinity sites on the cell surface. Once revealed (Grainger & Reckless (2003) Biochem. Pharmacol. 65: 1027-34), using conventional molecular screening tools (eg receptor binding assays), new, structurally unique BSCI groups It was impossible to search for. Using a functional screening tool (ie, to determine if library elements block cell migration in response to several different chemokines (not non-chemokines)), a very small library for BSCI activity Although it may be possible to screen directly, such a screening strategy becomes immediately impractical because of the labor intensive nature of functional screening assays (functional screening assays to identify inhibitors of cell migration). For a detailed review of technical difficulties to implement, see Frow et al. (2004) Med. Res. Rev. 24: 276-98). In addition, such functional screening is soluble in biologically compatible buffers and has low cytotoxicity (mostly (typically over 90%) with typical random libraries used for high-throughput drug screening. It can be applied only to compounds showing parameters) that exclude compounds of Briefly, such an approach is completely impractical to find a completely new class of BSCI from a random compound library.

  The ability to identify new, structurally unique BSCIs is important for the clinical development of this group of drugs. Existing BSCIs advance through clinical development, so it is unlikely that non-optimal properties will be identified. To date no BSCI has been administered to humans, and the risk of side effects or toxicity not identified to date remains high. Even worse, most of the side effects or toxicities that are manifested are class effects that are shared by compound classes with related structures. Therefore, unfortunately, all classes of compounds described to date for BSCI have been identified as a result of their structural similarity, thus increasing the risk of finding shared side effects.

  Here we describe three different receptors used by different groups of BSCIs. All three receptors are members of the GPCR group, all of which are highly expressed on leukocytes and in the brain. Surprisingly we also demonstrate that the reported BSCI acts with functional selectivity, regardless of the specific receptor being targeted. These results provide molecular screening examples useful in accordance with the present invention to identify structurally diverse BSCI groups that do not contain the same structural motifs as known compounds. The present invention is based in part on these results.

  The invention is defined in the appended claims and the following. In certain embodiments, one or more compounds or agents are screened for interaction with GPCR neuroreceptors expressed on one or more leukocyte subsets, followed by a second step. Methods are provided in which those compounds or substances that interact with the target receptor are screened for the ability to inhibit chemokine-induced leukocyte migration. Compounds or substances that inhibit chemokine-induced leukocyte migration are then tested for BSCI activity in vitro. Importantly, not all compounds or substances that are agonists for the target receptor have BSCI activity. Thus, the two steps of the present invention are necessary and sufficient to identify compounds having BSCI activity.

  Specific non-limiting examples and additional features of the present invention are described with reference to the following drawings.

The major BSCI structures described herein are shown, illustrating the interrelationship (and common structural motifs) shared by all of them. FIG. 2 shows the concept of functional selectivity at a single receptor (gray shape). FIG. 2 shows two known alternative transcripts (T # 1 and T # 2) obtained from the sstr2 locus as a result of alternative splicing of a single intron (darker square) in the protein coding frame. FIG. 6 is a graph showing the effect of increasing concentrations of the sstr2 antagonist BIM23627 (“I”) on the dose response of BSCIBN83253 (x axis, nM) and MCP-1 induced THP-1 cell migration (y axis,% inhibition). . BSCI (S) -3- (2 ′, 2′-dimethylpropionoylamino) -tetrahydropyridin-2-one dose response (“valerolactam”; x-axis, nM) and MCP-1 induction 2 is a graph showing the effect of increasing concentration of sstr2 antagonist BIM23627 (“I”) on sex THP-1 cell migration (y-axis,% inhibition). FIG. 5 is a graph showing the effect of co-administration (x-axis, mg / kg dose) of sstr2 antagonists BIM23627 and BSCIBN83250 on TNF-alpha production (y-axis,% inhibition) in response to a single intraperitoneal administration of bacterial endotoxin . Sstr2 antagonists BIM23627 and BSCI (S) -3- (2 ', 2'-dimethylpropionoylamino) for TNF-alpha production (y-axis,% inhibition) in response to a single intraperitoneal administration of bacterial endotoxin -Is a graph showing the effect of co-administration of tetrahydropyridin-2-one ("valerolactam"; x-axis, mg / kg dose). Co-administration (x-axis, mg / kg dose) of the sstr2 antagonist BIM23627 and the synthetic corticosteroid dexamethasone to TNF-alpha production (y-axis,% inhibition) in response to a single intraperitoneal administration of bacterial endotoxin It is a graph which shows an influence. PCR with (+) and without (-) reverse transcription ("RT") from mRNA samples prepared from THP-1 cells (left panel) and Panc-1 cells (right panel) used as positive controls Agarose gel stained with ethidium bromide and observed by UV light transmission revealing amplicons from the reaction. BN83250 is a labeled analogue ^{4 ', 5'- 3 [H]} - (S) -3- (2', 2'- dimethyl dodecanoyl) amino - caprolactam (4,5- ³ [H] -BN83250) The synthesis scheme of is shown. Compound (S, E) -3- (2 ′, 2′-dimethyldodec-4′-enoyl) amino-caprolactam (top) reacts with tritium gas in the presence of Pd catalyst to give 4,5- ³ [H ] -BN83250 (bottom) is formed. 4,5- ³ [H] -BN83250 (x-axis, nM) to THP-1 cells is a graph showing the specific binding (y-axis, cpm) of. Values are the mean (± SD) of triplicate measurements at each BN83250 concentration, minus non-specific binding (in the presence of excess unlabeled BN83250). The center line is the best fit when using a simple combined model and the outer line shows a 95% confidence limit. Various concentrations of unlabeled (S) -3- (2 ′, 2) for binding of 10 nM 4,5- [ ³ H] -BN83250 to THP-1 cells (y-axis, bound label [cpm]) 2 is a graph and histogram showing the effect of '-dimethylpropionoylamino) -tetrahydropyridin-2-one (“valerolactam”). Of pcDNA3.1 + (Invitrogen) with a 1,100 bp cDNA encoding human sstr2A (shown as “sstr2a”) cloned between the EcoRI (5 ′) and XhoI (3 ′) sites in the polylinker A plasmid map is shown. The effects of BSCI (BN83250) on SDF-1α-induced migration of parent Jurkat T cells (open circles, solid lines), Jurkat D3B cells expressing sstr2 (filled circles, solid lines), and THP-1 cells (dotted lines) are compared. In (A), the concentration of BN83250 is shown on the x-axis (nM) and% inhibition is shown on the y-axis. Frequency histograms showing the distribution of staining for each cell line are shown: (B) THP-1 cells (* = 375 median units of specific binding), (C) Jurkat D3B cells (* = 40 median units of specific binding) ), And (C) Jurkat T cells (* = one median unit of specific binding). In each frequency histogram, the x-axis shows Fl1-H and the y-axis shows the count. The black lines in the frequency histograms (B)-(D) indicate staining with PBS only; the light gray line indicates the primary antibody control and the medium gray line indicates the complete staining response. FIG. 5 shows the effect of various concentrations of somatostatin 14 (x axis, nM) on MCP-1 induced migration of THP-1 cells (y axis,% inhibition). The dotted line indicates complete inhibition, which is achieved by BSCI. Apparent affinity of sstr2 binding (x-axis; “Ka” at sstr2) for both a broad range of “classical” sstr2 agonists (A side) and “BSCI type” sstr2 agonists (or “somatotaxins”, B side) , NM) is a graph plotting the efficacy of inhibition of chemokine-induced migration (y-axis; ED50 vs. chemokine-induced migration, nM). 2 shows the structures of various novel beta-methionine tryptophan-based sstr2 agonists (L-799,976 and compounds AF) designed as candidate BSCIs for testing according to the methods of the present invention. 2 is a graph showing inhibition of chemokine MCP-1 induced THP-1 cell migration (y axis, shown as a percentage value) by Compound P (x axis, nM) according to one embodiment of the present invention. FIG. 5 is a graph showing inhibition of growth hormone (GH) release by compound P (x-axis, nM) (y-axis, shown as a percentage value). Compare levels of C-reactive protein (CRP; y-axis, ng / ml) as a surrogate marker of inflammation in serum samples from patients treated with Medrol® (A) or Compound P (B) This is a histogram. The column shows the CRP level before administration (a) and the last administration day (b). An asterisk indicates that p = 0.02.

  Both the BSCI peptide and aminolactam target receptors are disclosed as somatostatin type 2 receptor (sstr2) (see Examples below), and the target receptor for yohimbamide (eg, yohimban-16-amide) is α2- Because these are adrenergic receptors and the target receptors for lysergamides (eg L-II) are 5-HT2a, all acting with functional selectivity, chemokine-induced migration for all these receptors It is clear that there are functionally selective ligands that promote inhibition of Since all of these receptors are members of a larger class of GPCR neuroreceptors expressed on leukocytes, a similar function is seen for many if not all GPCR neuroreceptors expressed on leukocytes. It is reasonable to assume that ligands with selective selectivity are found. Accordingly, the methods of the invention include, in certain embodiments, screening one or more candidate compounds or substances for interaction with GPCR neuroreceptors expressed on leukocytes.

  In certain applications of the methods of the invention, one or more compounds to be screened are first on leukocytes by exposing cells expressing the selected receptor to a radioligand that can bind to the receptor. Tested for the ability to interact with the expressed GPCR neuroreceptor. For example, cells expressing the somatostatin receptor sstr2 are exposed to a ligand of sstr2 (eg, labeled somatostatin) in the presence and absence of the test compound. If the test compound interacts with the sstr2 receptor, the amount of somatostatin that specifically binds to the receptor will vary. Such receptor binding assays can be performed in a number of different ways to achieve the same goal: different ligands can be used (eg, sstr2-specific antagonist BIM23627; Tulipano et al. (2002) 143: 1218-24); different reporter systems can be used to visualize ligand binding (scintillation proximity assay and fluorescence polarization are two such examples); different from recombinant systems Cells (including cell membrane preparations or sstr2-expressing cells) can be used; different vehicles can be used to expose the cells to the test compound; different buffers to observe the specific binding of the ligand Conditions can be used, etc.

  In the second step of the above example, compounds that interact with the sstr2 receptor are screened for inhibition of chemokine-induced leukocyte migration. For example, leukocytes are placed in Transwell® migration assay plates in the presence of a gradient of chemokine MCP-1 and incubated for 2 hours at 37 ° C. in the presence or absence of the test compound (Frow et al. (2004) Med. Res. Rev. 24: 276-98). Next, at the end of this experiment, the number of migrated cells is measured using, for example, the vital dye MTT and interpolation into a standard curve. If the test compound inhibits chemokine-induced migration, few will migrate in the well receiving the compound. Such migration assays can be performed in many different ways to achieve the same goal: different migration chambers can be used (multiwell format Transwell® migration chamber, conventional boden chamber, in-gel migration Different chemokines may be used (SDF-1α, IL-8, MIP-1α, RANTES, etc.); different white blood cells may be applied to the chamber (freshly prepared peripheral blood neutrophils or Including lymphocytes, peritoneal macrophages, or cell lines such as THP-1 or Jurkat); different detection systems may be used to quantify the number of migrated cells, etc.

  Similarly, the methods of the invention can be applied to any other GPCR neuroreceptor expressed on leukocytes. Examples of such receptors include, but are not limited to, adrenergic receptors (including α2-adrenergic receptors), serotonin receptors (including 5HT2a receptors), somatostatin receptors (including sstr2), dopamine receptors , Muscarinic acetylcholine receptor, substance P / NPY receptor, vasopressin receptor, thyroid hormone receptor, cannabinoid receptor, cholecystokinin receptor, opioid receptor, adenosine receptor, glucagon receptor, PACAP / VIP receptor , Melatonin receptors, bradykinin receptors, calcitonin receptors, and melanocortin receptors.

  Preferably, the method of the present invention is applied to an adrenergic receptor, a serotonin receptor, or a somatostatin receptor. More preferably, the method of the present invention is applied to α2-adrenergic receptor, 5HT2a receptor, or sstr2 receptor.

  If this method is applied to somatostatin receptors (including sstr2), the candidate compound or substance may exclude somatostatin.

  The methods of the present invention can be readily applied to screen a library of compounds to identify those most likely to have BSCI activity. Such libraries can be random libraries (eg, large libraries of structurally diverse compounds used by many pharmaceutical companies for high-throughput screening) or directed libraries (eg, , A library synthesized by combinatorial chemistry applied to a limited number of precursor compounds). Some directional libraries are likely to contain BSCI compounds (or conventional agonists for selected receptors), so such libraries are screened using the methods of the present invention, It can be particularly important. For example, the G protein agonist enriched library (GAEL) library that we have already disclosed (see WO2006 / 085096) is a novel class of agonists that are apparently acting as a special class of agonists in certain GPCRs. It can be screened productively according to the method of the invention. Use of a directional library (eg, GAEL) can reduce the number of compounds that need to be screened to identify compounds having candidate BSCI activity according to the criteria disclosed herein. However, the method of the present invention is sufficiently high throughput, which is successfully applied to random libraries, and such applications can be encompassed by the present invention.

  Optionally, the activity of BSCI as a “classical” agonist for the selected receptor is also measured. Preferably, a compound or substance identified according to the method of the present invention has both types of functional assays (ie cell migration assays for BSCI activity and classical agonist activity for selected receptors). It is an inhibitor of chemokine-induced leukocyte migration that is active in a functional assay) but more potent (ie, acts at a lower concentration) than its potency as a classic receptor agonist.

  That is, the optimal BSCI can be identified as a compound having all of the following three properties according to the method of the present invention:

(I) The compound interacts with a selected GPCR neuroreceptor expressed on leukocytes with an affinity of at least 10 μM (ie, more than half of the sstr2 receptors present when the compound is present at 10 μM). Having compounds bound to them);
(Ii) the compound inhibits chemokine-induced leukocyte migration with a potency of at least 1 μM (ie, the extent of cell migration is inhibited by at least 50% when the compound is present at 1 μM); and (iii) The potency of a compound in one or more assays of classical sstr2 agonist activity at a selected receptor is lower than that for inhibition of leukocyte migration.

  More preferably, the compound may interact with a selected receptor with an affinity of at least 1 μM; more preferably, the compound may inhibit chemokine-induced leukocyte migration with an efficacy of at least 100 nM; more preferably leukocytes The potency of a compound for migration can be more than 10 times higher than the affinity of interaction with the selected receptor or the potency of classical agonist activity.

  More preferably, the compound is capable of inhibiting chemokine-induced leukocyte migration with a potency of at least 10 nM; more preferably, the potency of the compound against leukocyte migration is an affinity for interaction with a selected receptor or a classical agonist. It can be more than 50 times higher than the potency of the activity.

  It is clear that the methods of the present invention can distinguish between conventional agonists for these neuroreceptors (including but not limited to natural ligands) and compounds having BSCI activity. While compounds that are conventional agonists can inhibit chemokine-induced leukocyte migration, they inhibit with similar or lower potency with an affinity for interaction with specific neuroreceptors.

  Similarly, conventional agonists for these receptors have conventional effects via the target receptor (eg, inhibition of growth hormone release, or blood pressure regulation, modulation of insulin release, eg, depending on the particular receptor being tested). It only shows an effect on chemokine-induced leukocyte migration at the same concentration as shown. Thus, although conventional agonists for these neuroreceptors have anti-inflammatory activity at certain doses, such compounds also show the action of natural ligands and act through the target receptor at the same compound dose. To do. It should be emphasized that an important advantage of the method of the present invention is not only to identify substances that have anti-inflammatory properties, but also to identify superior subclass substances whose anti-inflammatory properties exceed side effects. It is. Specifically, the substances identified have greater anti-inflammatory efficacy than most classical agonists for GPCR neuroreceptors expressed on leukocytes (most of the GPCR neuroreceptors expressed on leukocytes). Properties not shared by known agonists).

  Peptide BSCI and aminolactam BSCI are special (and not yet identified) subsets of sstr2 agonists. Although these BSCIs and conventional sstr2 agonists share a common molecular target (sstr2 receptor), the two groups of substances cause different results for binding to the receptor. This concept is described in detail elsewhere for other receptors (Urban et al (2007) J. Pharmacol. Exp. Ther. 320: 1-13) and is called functional selectivity. It is. A useful parable is to consider the receptor (in this case sstr2) as a piano keyboard: many different hand positions (corresponding to different ligands) can interact with the keyboard (corresponding to productivity binding). , Each producing a different chord sound (corresponding to a different functional result from the receptor, eg a different second messenger is produced). The concept of this functional selectivity is shown in FIG.

  Yohimbamide BSCI (including yohimbane-16-amide) is a special (and yet unidentified) subset of α2-adrenergic receptor agonists. Although these BSCIs and traditional α2-adrenergic receptor gonists (eg reserpine) share molecular targets, the two groups of substances differ in their binding to receptors (Urban et al (2007) J. Pharmacol Exp. Ther. 320: 1-13) (a phenomenon called functional selectivity). Yohimbamide potently inhibits chemokine-induced leukocyte migration but does not affect blood pressure; reserpine strongly reduces blood pressure but does not affect chemokine-induced leukocyte migration.

  Lysergamide BSCI (including L-II) is a special (and yet unidentified) subset of 5HT2a agonists. Although these BSCIs and conventional 5HT2a agonists (eg TCB2; McLean et al. (2006) J. Med. Chem. 49: 5793-803) share a common molecular target (5HT2a), the two groups of substances are Causes different results for receptor binding (Urban et al (2007) J. Pharmacol. Exp. Ther. 320: 1-13) (a phenomenon called functional selectivity). Lysergamide potently inhibits chemokine-induced leukocyte migration but does not affect hallucinogenicity (a characteristic neck twist in rodents); TCB2 is a characteristic head twist of hallucinogenic activity ), But does not affect chemokine-induced leukocyte migration.

  The knowledge disclosed herein that BSCI is a special class of function-selective agonists for the various GPCR neuroreceptors known to be expressed on leukocytes is based on the fact that compounds having BSCI activity are pre- Allows identification without structural information (ie, without pharmacophore information common to already described BSCI compounds).

  Such compounds can be identified, for example, as follows according to the method of the present invention. The first step is screened for interaction with selected GPCR neuroreceptors known to be expressed on leukocytes. BSCI is then distinguished from conventional agonists for its receptor by its ability to inhibit chemokine-induced leukocyte migration with more potent potency than classical agonist activity for the conventional target or its receptor. Optionally, in a fourth step, structural motifs can be identified that define a new class of BSCI that is structurally unrelated to those already disclosed.

  The first step involves screening for interactions with selected GPCR neuroreceptors known to be expressed on leukocytes. To satisfy this definition, the receptor is (a) a member of the GPCR superfamily according to structural and operational criteria known in the art; (b) expressed in the central nervous system at a functionally significant level. And (c) must be known to be expressed at a functionally significant level on leukocytes or a subset of leukocytes. The requirement that the receptor selected for the practice of the present invention is a member of the GPCR superfamily and is expressed in the central nervous system is that BSCI functions at all three different receptors that meet this definition. Based on the finding that it is a selective ligand. The requirement that this receptor is expressed on leukocytes is based on the desire that the selected substance inhibits chemokine-induced leukocyte migration.

  It is contemplated that the selection of one or more receptors used for the specific implementation of the method of the invention is based primarily on the expression pattern of the receptor across different leukocyte subsets. For example, if a substance is needed that selectively suppresses neutrophil migration in response to chemokines, GPCR neuroreceptors that are selectively expressed on neutrophils (as compared to other leukocytes) can be selected. Similarly, if a substance that selectively inhibits mast cell migration is required, GPCR neuroreceptors expressed on mast cells can be selected. Alternatively, changes in the expression pattern of GPCR neuroreceptors during disease can be stimuli to select specific receptors; substances that are selectively expressed on eosinophils in asthmatic lungs are used in asthma Could be an ideal target for identifying anti-inflammatory agents. Thus, the need for a solid or group applying the method of the present invention can readily provide a basis for selecting an appropriate GPCR neuroreceptor for the application of the present invention, It is in no way limited to a particular approach for selecting GPCR neuroreceptors to be made.

  The second and third steps involve performing a functional assay for (1) inhibition of chemokine-induced leukocyte migration and (2) for classical agonist activity for selected receptors. Both of these two steps are necessary to clearly define substances with functional selectivity for BSCI activity mediated by GPCR neuroreceptors. However, these two steps can be performed in sequence or essentially simultaneously. It is important to use leukocyte populations for functional assays of BSCI activity that are known to express the specific target GPCR neuroreceptor selected in the first step. Failure to do this results in false negative results from this method, in that all substances that may have functional selectivity for BSCI activity do not express the target receptor and do not interact with it. This is because it has no effect on inducible migration.

  A fourth step is optionally applied to identify common structural motifs associated with functionally selective BSCI activity. Typically, such a process involves a compound newly identified as BSCI according to the method of the present invention, such that a repetitive structural element is likely to match the motif required to confer BSCI activity. Comparing the structural elements between. Next, we empirically demonstrate the importance of such motifs, for example by synthesizing various molecules with related structures and proving that the presence of the motif is related to the presence of BSCI activity. be able to. As a result, even if only a single member of the class has been uniquely identified through screening for interaction with GPCR neuroreceptors expressed on leukocytes, the method of the present invention provides a class of BSCI compounds. Can be used to define the whole (members of each class are related to each other in structure).

  That is, it is contemplated that the BSCI compounds identified according to the methods of the present invention serve as structural leads for the synthesis and characterization of novel and structurally related BSCI families. Consequently, such structural derivatives of the first hit identified by the method of the present invention are not directly identified by application of the method of the present invention, but the critical steps in their identification depend on the method of the present invention. Thus, indirect identification of relevant BSCI groups in this way is contemplated according to this aspect of the invention.

  When a library is screened according to the methods of the present invention, the library elements are individual compounds (or at least substantially pure compounds, where the target compound comprises at least 25% of the material present and higher There may be no other compounds at the level), or the library elements may be a mixture of compounds. Where the element is a mixture of compounds, the components of the mixture can be known or can be predicted (as in many combinatorial libraries) or unknown (eg, an isolate of a natural product) possible.

  If some or all of the library elements that are screened according to the method of the invention are a mixture (having a known or unknown composition), they are expressed on leukocytes after the first step of the method of the invention After a specific compound is isolated and identified from the mixture responsible for the interaction with the selected GPCR neuroreceptor, the second step (assessment of the compound's effect on chemokine-induced leukocyte migration) is applied It is contemplated. If such an isolation step is not included in the method, the method selects one compound in the mixture when a single compound in the mixture has (if necessary) BSCI activity. It would be indistinguishable from a case where a second unrelated compound interacts with a neuroreceptor and inhibited leukocyte migration by a mechanism unrelated to BSCI activity.

  For example, the method of the invention is applied to a library element consisting of a mixture of somatostatin-14 (a conventional sstr2 agonist but not BSCI) and sodium azide (a cytotoxin that inhibits cell migration but not BSCI). The mixture exhibited a strong interaction with sstr2 and inhibited chemokine-induced leukocyte migration with a potency higher than predicted by affinity at sstr2 (depending on the relative concentration of azide present). obtain. Such a mixture can be incorrectly identified as BSCI. However, if the component of the mixture responsible for interaction with sstr2 (somatostatin in this example) as described above is isolated from the mixture prior to applying the second step of assaying for effects on chemokine-induced migration. If so, this method can correctly identify the mixture as lacking BSCI activity.

  In principle, the steps of the method of the invention can be applied in any order (eg, measuring the potency of a compound under test as an inhibitor of chemokine-induced migration and then expressing a GPCR expressed on leukocytes. Although it is in principle possible to measure the affinity of a compound's interaction (if any) with a neuroreceptor), such methods lack the advantages of preferred embodiments of the invention . In these situations, functional assays cannot be used to screen random libraries (due to the presence of toxic and insoluble compounds as well as due to the resources of such screening). Accordingly, in the preferred invention, screening for interaction with GPCR neuroreceptors expressed on leukocytes is performed as the first step, followed by functional assays for inhibition of chemokine-induced leukocyte migration. Screening to assess interactions with GPCR neuroreceptors expressed on leukocytes (example of molecular screening) is screened rather than measuring activity against chemokine-induced leukocyte migration (example of functional screening) This process sequence reflects the optimal use of available resources since much less resources are required per compound.

  Application of the methods of the invention is contemplated to include virtual screening as well as actual or physical screening. That is, some (or all) of the information necessary to identify a compound as a candidate BSCI according to the method of the present invention is already available. For example, a library of compounds has already been screened for interaction with various GPCR neuroreceptors expressed on leukocytes prior to the present disclosure, and the results may be stored (eg, in a computer hard disk). I don't know. Thus, a subset of compounds from the library can be selected as inhibitors of chemokine-induced migration in leukocytes and then tested for efficacy and identified as candidate BSCIs according to the methods of the invention. It is clear that such a procedure is an application of the method disclosed herein and relies on the criteria disclosed to identify candidate BSCIs. Similarly, for a series of compounds, the affinity of each interaction with one or more GPCR neuroreceptors expressed on leukocytes is already known and separately for inhibition of chemokine-induced leukocyte recruitment. If the potency of a compound is already known, the combination of existing information to identify which compound is a candidate BSCI can be an application of the method of the invention, even if no new experimentation is required.

  In particular, if one or more steps of the present invention do not require new experimentation, the steps can be performed in any order (ie, need not be in the preferred order described herein).

  A number of anti-inflammatory activities attributed to agonists for GPCR neuroreceptors expressed on leukocytes have been described (eg, Paran & Paran (2003) Curr. Opin. Invetig. Drugs 4: 578-82; Sener et al. (2005) Peptides 26: 493-9; Gonzales-Rey et al. (2007) Ann. Rheum. Dis. 66: 582-8 and references therein; Armani et al. (2007) J. Leukoc Biol. 81: 845-55; Cury et al. (2008) Dig. Dis. Sci. 53: 2516-20, which are for a single GPCR neuroreceptor, sstr2, expressed on leukocytes. Similar examples exist for many other GPCR neuroreceptors). Thus, the novelty and inventive step of the present invention is the discovery that ligands that act via GPCR neuroreceptors expressed on leukocytes can have clinically useful anti-inflammatory effects (only a single step). There is no discovery teaching the invention to have, ie screening for interaction with GPCR neuroreceptors expressed on leukocytes to identify anti-inflammatory agents that may be useful. Instead, the present invention provides that some of the agonists (and possibly very small ratios) for GPCR neuroreceptors expressed on leukocytes are substantially classical agonist activity (eg, suppression of growth hormone secretion). Instead of blood pressure or hallucinogenic regulation), it relates to the unexpected finding that it has anti-inflammatory activity.

  This type of functional selectivity (different ligands bind to and activate the same receptor but induce significantly different functional results) is known in the pharmaceutical literature (for review see Urban et al. al (2007) J. Pharmacol. Exp. Ther. 320: 1-13), but there is little firm literature. Thus, it may not be possible to estimate whether a functionally selective ligand is present for a receptor or for a pair of functions. Therefore, prior to our disclosure, those skilled in the art reasonably concluded that the anti-inflammatory activity associated with GPCR neuroreceptor activation is always accompanied by a classic agonist response, and the discovery of new drugs with anti-inflammatory activity Limited the usefulness of GPCR neuroreceptors as targets for use. Our disclosure shows that functional selectivity exists not at only one GPCR neuroreceptor, but at three different GPCR neuroreceptors, and in addition, in each of these cases little or no agonist-like activity. It is clearly shown that it can exhibit substantial anti-inflammatory activity. Based on the information available prior to our disclosure, even those skilled in the art (including the present inventors) did not anticipate this finding, nor was it possible or of course presumed to be probable .

  The present invention provides a method for the unambiguous identification of substances that exhibit functional selectivity and promote BSCI activity over classical agonist activity for selected GPCR neuroreceptors. In order to effectively practice the present invention, it is not necessary to understand the molecular details of how this functional selectivity is achieved nor the structural properties between the ligands that define the desired result. The information provided in this disclosure is both necessary and sufficient to teach those skilled in the art to select functionally selective substances that would be useful from a pool of candidates that are inherently equally promising. However, it may be useful to illustrate the extent to which this finding is unpredictable and how functional selectivity must be achieved.

  When a ligand binds to a receptor, there are multiple non-covalent interactions between the ligand molecule and the receptor protein (including but not limited to van der Waals forces, hydrogen bonds, dipole interactions, and Including a salt bridge; see FIG. 2). These interactions are involved in both the affinity of the ligand-receptor interaction (thus the selectivity of binding to the target receptor but not other proteins) and the signaling response to ligand binding. In natural ligands, these interactions cause a change in the conformation of the receptor protein (the minimum energy conformation of the receptor: ligand complex is distinct from the minimum energy conformation of the receptor and ligand when separated) Different). This conformational change converts the signal from ligand binding out of the cell to an intracellular signaling cascade. This transformation is direct (as in the case of receptors with endogenous kinase activity) or indirect (as in the case of G protein coupled receptors, eg sstr2), and the conformational change is 1 or Promotes the association of two or more additional proteins, which transduce the signal. A substance that binds to the receptor and reproduces the conformational change in the receptor seen with a natural ligand can be an agonist for the receptor (FIG. 2A). Agents that bind to the receptor without inducing these changes can be antagonists (assuming that this prevents binding to the natural ligand; FIG. 2B). However, under unusual circumstances, a third possibility arises: the substance binds and induces a very different set of conformational changes that cause qualitative rather than quantitative changes in the nature of the signal, This is then converted (FIGS. 2C and D). It is this third unusual scenario that dictates the pharmacological phenomenon of functional selectivity (and to some extent super agonists and inverse agonists).

  This molecular scenario may be easier to understand when compared to music. In this model, the piano keyboard is an example of a receptor and the pianist's hand is a ligand. Under normal conditions, hand action (a specific conformation) strikes a specific chord. This “signal” is the agonist response to the receptor. Closing the piano lid prevents the interaction between the ligand and the receptor without inducing a conformational change at the keyboard position, so no chord (agonist signal) is heard. The piano lid is an analog of an antagonist. Replacing a pianist with a child who does not know how to play the keyboard (an example of a compound randomly selected from the library) will give a mysterious response rather than a musical chord in almost all cases. However, in rare cases, you may encounter a genius and play a beautiful chord that is completely different from the pianist. The same receptor now responds with functional selectivity: different ligands elicit qualitatively different responses from the same receptor.

  From the above, it will be clear that there is no way to predict functional outcome from different ligands that bind to the same receptor. In fact, for some receptors it is impossible to even predict what signals are possible (in the sense that no matter how you press the keys, you cannot make a violin sound from the piano keyboard). ). Performing experimental screening based on the information disclosed after accidental discovery is a method of discovering substances with the desired functional selectivity (eg, the BSCI described herein).

  It is clear that the present invention consists of three or more steps, each single step being a method known in the art. For example, to replicate the known action of natural ligands or to search for substances that interact with GPCR neuroreceptors expressed on leukocytes in order to block known actions of natural ligands. It is clear that the first step is used in this case. Similarly, we and other researchers have disclosed the use of functional assays for chemokine-induced leukocyte migration with the goal of characterizing substances for BSCI activity. However, prior to this disclosure, quite different results (ability to screen for compounds with BSCI activity in random libraries; most somatostatin receptor interactors are not BSCIs, most elements in random libraries are The ability cannot be achieved using any of the steps alone because it is not compatible with a functional assay for BSCI activity, and these two methods are combined and performed sequentially There was no possible rationale for. However, the information that BSCI interacts with the GPCR neuroreceptor expressed on leukocytes and acts by functional selectivity, which is disclosed for the first time in this document, suggests that these two unrelated processes Can be combined into a new method with unexpected utility to enable the present invention. A good analogy to the method of the invention is the composition of a new peptide: the peptide consists of (almost all) the same amino acid building blocks and is bound in the same way. However, because there are so many combinations, the identification of a specific combination (new peptide sequence) with a specific function is considered inventive. Similarly, there are so many possible receptors that BSCI can act on (even if the search is limited to those known to be able to mediate anti-inflammatory activity in vivo) and molecular screening for each receptor, Trying to combine with functional screening for BSCI activity was not feasible. The present invention provides a method for identifying BSCIs from random libraries, thereby realizing this realization for useful combinations that are unexpected and particularly superior to many other combinations that should have been equally possible at first. Overcome impossible searches.

  The methods described herein are equally applicable to homologs of GPCR neuroreceptors of other species, including but not limited to mice, rats, dogs, and primates, and non-mammalian species such as birds. If a non-human homolog of a GPCR neuroreceptor is used to identify a compound according to the present invention intended for human use (eg, as an anti-inflammatory drug), it is identified using a non-human receptor Additional steps may be required to check the material being processed for functional selectivity for human receptors. Preferably, however, if the BSCI activity in the human system is the desired result of the screening procedure, the method of the invention should be applied to human GPCR neuroreceptors expressed on leukocytes. Of course, it is most preferred to use GPCR neuroreceptor analogs from one or more target species if the desired result is BSCI activity (eg, veterinary drug) in the non-human system of the method. possible.

  It is contemplated that the methods of the invention can be applied using incomplete fragments of a receptor, provided that the receptor fragment retains the ability to bind to the natural ligand of the full-length receptor or another ligand. Is done. Typically such functional fragments contain the majority of the protein sequence, while those not involved in ligand binding (eg to improve expression or stability) have an impact on the application of the invention. Can be deleted without giving.

  Measure test substance binding to GPCR neuroreceptor either indirectly (by competition for binding to another previously labeled ligand for the receptor) or directly (test substance itself has label) Obviously we can do it. The scope of the present invention is in no way limited to the methods used to detect the interaction between the test substance and the receptor.

  The functional assay used to detect the BSCI activity of the test substance may be in vitro (eg, using Transwell® migration assay) or in vivo (eg, after intraperitoneal injection of chemokine, in response to leukocyte mobilization. It will be clear that it can also be carried out (using evaluation). In addition, alternative markers of leukocyte recruitment (both in vitro and in vivo) can be used for assessment of BSCI activity. For example, leukocytes are known to express the cytokine TNF-alpha (especially when activated). Consequently, measured levels of TNF-alpha in vitro or in vivo are an alternative method for assessing leukocyte recruitment and thus a method for measuring BSCI activity. The scope of the present invention is in no way limited to the methods used to detect BSCI activity associated with a particular test compound.

  The functional assay used to detect the classical agonist activity of the test substance is either in vitro (eg, by measuring GH release from pituitary cells for a somatostatin receptor agonist) or in vivo (eg, a somatostatin receptor agonist). It will be clear that this can also be done by measuring the GH level in the serum. In addition, alternative markers can be used to estimate classical sstr2 agonist activity. Using an example of a somatostatin receptor agonist again, so that IGF-I levels are measured as an indicator of GH levels, and thus as an estimate of classical somatostatin receptor agonist activity, GH is insulin-like growth factor I ( Control the level of IGF-I). The scope of the present invention is in no way limited to the methods used to detect classical agonist activity associated with a particular test compound.

  Optionally, an additional step is performed to allow identification of a new structural class of BSCI based on the presence of one or more structural features of the compound selected during the previous step of the method of the invention. It may be added to the method of the present invention. The identification of such structural motifs may be experimental or theoretical in nature in order to identify hits from libraries (whether random libraries or those selected from candidates) through the application of the method of the invention. A far wider array of BSCIs is possible than only sought compounds. For example, after screening a random library for binding to a GPCR neuroreceptor expressed on leukocytes (first step of the invention), measurement of inhibition of chemokine-induced leukocyte migration (second step) and selected Several new compounds can be selected according to the method of the present invention by occasional measurement of the potency of the receptor against the “classical” target. Two or more of these selected compounds may share a common structural motif (eg, the presence of a 3-substituted cis-decalin ring system). Thus, in any additional step, the presence of a 3-substituted cis-decalin ring system imparts the desired properties to the compound, and additional 3-substituted cis-decalin containing molecules can be synthesized with appropriate predictions, at least a portion of which Obviously, it can have the desired properties. Under such circumstances, the definition of a new structural motif associated with BSCI activity or “somatotaxin” activity may constitute an application of the method of the invention.

  Thus, the method of the invention comprises at least three steps (measurement of interaction with GPCR neuroreceptors expressed on leukocytes, followed by functional assays for BSCI activity, and “classical” for selected receptors). "Functional assay for agonist activity). A final step may optionally be added (defining a structural motif associated with BSCI activity). The advantage of this final step is that it allows the selection of compounds with the desired properties by structural homology rather than assay-based screening. Such “hit to lead” optimization programs are known in the art and facilitate the application of the method of the present invention to the discovery of new substances suitable for use as human drugs. To do. However, depending on the particular application, the resource problem of performing this optional final step may be greater than the benefit of expanding the range of compounds identified.

  As used herein, the terms “compound” and “agent” are generally considered synonymous and are thus interchangeable unless the context clearly indicates otherwise.

  The term “broad-spectrum chemokine inhibitor” or “BSCI” usually gives a signal that induces migration through different receptors, but is mostly for cell migration induced by non-chemokine chemoattractants. Or means a compound or substance capable of inhibiting cell migration in response to a wide range of chemokines that have no effect. This is an operational definition and it is clear that different compounds or substances can be BSCI as a result of completely different molecular mechanisms. However, the present invention is only relevant to the identification of BSCIs acting through sstr2 or related receptors identified in the present invention, an important and extensively studied subclass of BSCI.

  The term “BSCI activity” refers to a number of agonists acting at GPCR neuroreceptors expressed on leukocytes in vitro or in vivo to chemokine-induced cell migration as well as related in vivo immunological changes (eg, Figure 2 shows the effect on T helper (CD4 +) cell cytokine secretion profile readjustment).

  The term “classical agonist activity” means that a cell that expresses a sufficient level of a GPCR neuroreceptor has any known agonist compound for that receptor (the natural one or more of the receptor). A group of cellular responses known to occur, possibly in different cell types, when exposed to (including ligands). For example, an example of classical agonist activity for the sstr2 receptor would be inhibition of GH release by pituitary cells, inhibition of insulin release by pancreatic cells (Olias et al. (2004) J. Neurochem. 89: 1057). However, a wide range of other effects of sstr2 agonists acting on various cells have also been described and these (except for the “BSCI activity” mentioned above) also include classical sstr2 agonist activity (Olias et al. (2004 ) J. Neurochem. 89: 1057). Thus, classical agonist activity is defined as all known activity of an agonist for a selected GPCR neuroreceptor, excluding BSCI activity, and thus all known activity of an agonist is encompassed by this definition.

  The term “sstr2” is encoded by the sstr2 gene (HGNC ID: 11331; complete gene sequence shown in SEQ ID NO: 1) on the human chromosome 17q24, and encodes the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing described below. Figure 2 shows the human type 2 somatostatin receptor.

  Unless otherwise stated, the term sstr2 is intended to encompass the product of any alternatively spliced or differentially processed mRNA resulting from transcription of the locus set forth in SEQ ID NO: 1. For the avoidance of doubt, the term sstr2 encompasses one or both of the sstr2a and sstr2b proteins resulting from alternative splicing of sstr2 mRNA with a single intron located in the protein coding frame. Other species of analogs or homologs are also encompassed by this term unless they are nonsense in a particular context.

  The term “somatostatin receptor” refers to any receptor (whether or not currently known) to which somatostatin binds with high affinity (typically 1 μM or less). For the avoidance of doubt, this includes sstr1, sstr2, sstr3, sstr4, and sstr5. Other species of analogs or homologues are also encompassed by this term unless they are nonsense in a particular context.

  The term “somatostatin receptor” further includes at least 50% identity, eg, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, with the amino acid sequence of human sstr2 (described above), Polypeptides having amino acid sequences with 90%, 95% identity, or at least 96%, 97%, 98%, or even 99% identity are included. The polypeptide preferably has the somatostatin receptor activity described above.

  Sequence identity between amino acid sequences can be determined by comparing sequence alignments. When an equivalent position in the compared sequences is occupied by the same amino acid, the molecules are identical at that position. Scoring the alignment as a percent identity is a function of the number of identical amino acids or bases at positions shared by the compared sequences. When comparing sequences, optimal alignment may require introducing gaps into one or more sequences to allow for possible insertions or deletions in the sequences. A sequence comparison method has a higher score for the same number of identical molecules in the compared sequences, with as few gaps as possible reflecting the higher association between the two comparison sequences, than with those with many gaps Use a gap penalty. The calculation of the maximum percent identity involves creating an optimal alignment taking into account gap penalties.

  Appropriate computer programs for performing sequence comparisons are available in the commercial and public sectors. Examples include MatGat (Campanella et al., 2003, BMC Bioinformatics 4: 29; program available from http://bitincka.com/ledion/matgat), Gap (Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443-453), FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410; program available from http://www.ebi.ac.uk/fasta), Clustal W 2.0 and X 2.0 (Larkin et al., 2007, Bioinformatics 23: 2947-2948; a program available from http://www.ebi.ac.uk/tools/clustalw2), and EMBOSS Pairwise Alignment Algorithms (Needleman & Wunsch, 1970 , supra; Kruskal, 1983, In: Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Sankoff & Kruskal (eds), pp 1-44, Addison Wesley; http://www.ebi.ac. available from uk / tools / emboss / align). All programs are used with default parameters.

  For example, sequence comparison is performed using the “needle” method of EMBOSS Pairwise Alignment Algorithms, which determines the optimal alignment (including gaps) of two sequences when considered over their entire length and is identical Provide a sex percent score. The default parameters for amino acid sequence comparison (“protein molecule” option) are: gap extension penalty: 0.5, gap opening penalty: 10.0, matrix: Blossum 62. The default parameters for nucleotide sequence comparison ("DNA molecule" option) are: gap extension penalty: 0.5, gap opening penalty: 10.0, matrix: DNAfull.

  The term “somatostatin” refers to any product of the gene encoding somatostatin (encoded by the sst gene on chromosome 3q28 (HGNC ID: 11329) in humans), which is a controlled proteolysis of the gene product Contains the fragments produced by Unless otherwise stated, the term somatostatin (or sst) encompasses any fragment (including but not limited to sst-14 and sst-28) produced by proteolysis assigned a biological function. Is intended. Other species of analogs or homologues are also encompassed by this term unless they are nonsense in a particular context.

  The term “leukocytes” refers to any or all leukocyte populations in the blood, as well as progeny of these cells in bone marrow or secondary lymphoid organs (eg, spleen), and progeny of these cells in tissues (eg, , Osteoclasts, Kupfer cells, microglial cells, mast cells, dendritic cells, etc.). Examples of leukocytes include monocytes, lymphocytes (including both B and T lymphocytes, killer T cells, helper T cells, regulatory T cells, and natural killer cells), and granulocytes (neutrophils, Basophils, eosinophils).

  The term “GPCR neuroreceptor” refers to a G protein-coupled receptor (GPCR) expressed at a functionally relevant level on cells of the central nervous system, including neurons, astrocytes, and microglia. Includes any member of the superfamily. G protein-coupled receptors are a well-defined class to clearly determine whether a receptor is a member of the GPCR superfamily based on its sequence and structure and on its signaling mechanism. Methods exist in the art. For example, GPCRs are typically identifiable, with seven transmembrane domains (each in the region of 12-20 hydrophobic amino acid residues, with one or more charged residues on either end) Is characterized by the presence of GPCRs are also characterized by their ability to interact with and activate members of the group of GTP binding proteins (referred to as G proteins). Examples of GPCR neuroreceptors include, but are not limited to, the following groups: adrenergic receptors (including α2-adrenergic receptors), serotonin receptors (including 5HT2a receptors), somatostatin receptors (sstr2 Dopamine receptor, muscarinic acetylcholine receptor, substance P / NPY receptor, vasopressin receptor, thyroid hormone receptor, cannabinoid receptor, cholecystokinin receptor, opioid receptor, adenosine receptor, glucagon receptor , PACAP / VIP receptor, melatonin receptor, bradykinin receptor, calcitonin receptor, and melanocortin receptor.

  The term “GPCR neuroreceptor expressed on leukocytes” refers to a subset of all GPCR neuroreceptors in which the receptors are expressed at a functionally significant level by at least one subset of leukocytes or leukocytes. Although the complete complement of GPCRs expressed on all leukocyte subsets is currently unknown, the term is used in the present invention since they are known to be expressed on at least one subset of leukocytes. All GPCR neuroreceptors selected for, as well as GPCR neuroreceptors selected based on screening of expression patterns in leukocytes (which were previously not known to be expressed on leukocytes ) Is used to explicitly include. GPCR neuroreceptors that are not described as present on leukocytes and that have not been selected by examining leukocyte-based expression cannot be included in this definition.

  The term “potency” is used to describe the concentration of a particular substance required to have a particular action. Typically, potency is the concentration of compound required to inhibit the endpoint under test by 50% (referred to as ED50 or IC50). Thus, an ED50 or IC50 at a low concentration indicates a compound with high potency. The potency is completely different from the “power” (described below) which shows the maximum effect of the compound on the endpoint under test at the concentration tested. The compounds are highly potent but have low power (eg, act on chemokine-induced migration with an ED50 of 0.05 nM, but inhibit migration only 40% at very high concentrations) or low potency (Eg, ED50 is 10 mM, but inhibits endpoints 100% at concentrations higher than 100 μM).

  The term “power”, when applied to a pharmacological assay, describes the maximum effect of a particular substance on the endpoint under test obtained by selecting the optimal concentration of that substance in that assay. Used for. The optimal concentration is typically the highest concentration tested, but in some instances (eg, when a bell-shaped dose response is commonly observed, eg, chemotactic response to chemokines) May have a maximum effect on the endpoint under test. The output is independent of potency, and a compound with low potency may have a high output or vice versa.

  The term “about” refers to the spacing around the considered value. As used in this patent application, “about X” is (10% of X−X) to (10% of X + X), preferably (5% of X−X) to (5% of X + X). Is the interval.

  The use of a numerical range in this description means that all individual integers within that range and all combinations of upper and lower numbers within the widest range of a range are included within the scope of the invention. Clearly plan.

  In this specification, the term “comprising” should be read to mean both comprising and consisting of. Conversely, the term “a pharmaceutical composition comprising a compound as an active ingredient” when the invention is used is a composition in which the active ingredient is defined as a composition in which the other active ingredient may be present. Is meant to encompass both.

  The following abbreviations, when used, refer to the 20 naturally occurring proteinogenic amino acids, even if isolated as part of a polypeptide or other amide-containing compound as appropriate from the context.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates.

  Specific non-limiting examples and additional features of the present invention are described with reference to the following drawings.

Several figures are described in more detail.
FIG. 1 shows NR58-3.14.3 (“A”), a cyclic retroinverso analog of the sequence obtained from peptide 3 (not shown). The tripeptide motif WxQ has been identified as the major pharmacophore, and tripeptides such as WVQ (“B”) have BSCI activity (although less potent). Yohinban-16-amide (“C”) and NR58,4 (“D”) have been identified as structural analogs of the WxQ tripeptide. Then BN83253 (“E”), BN83250 (“F”), and (S) -3- (2 ′, 2′-dimethylpropionoylamino) -tetrahydropyridin-2-one (“G”) Have been identified as stable imide analogs such as NR58,4. Lysergic acid asparagine and glutamine amide (eg L-II) have been identified as structural homologues of yohimbane-16-amide.

  In FIG. 2, panel A shows that the natural ligand interacts with the receptor and generates multiple responses (arrows), which are interpreted as intracellular signals or functional consequences. In panel B, the antagonist binds to the receptor and prevents the natural ligand from binding, but does not react with the receptor moiety necessary to induce active signaling. In panel C, a function-selective agonist binds to the receptor to preferentially activate one response (thick arrow) over another response (thin arrow). In panel D, different agonists with the opposite functional selectivity are illustrated.

  Referring to FIG. 3, in transcript A (“T # 1”), the intron has not been removed by splicing and the translation stop codon gives a truncated protein (referred to as sstr2a). In transcript B ("T # 2"), the intron has been removed by splicing, translation proceeds to the second exon and alternative translation termination code, and the sstr2b protein (which differs from sstr2a only with a short C-terminal tail) give. The positions of the PCR primers used to unambiguously identify the sstr2 transcript are shown as (a) for the sstr2 primer set and (b) for the sstr2A primer set. The amplicon sizes obtained from these two different transcripts are 398 bp (T # 1, sstr2 primer set), 87 bp (T # 1, sstr2A primer set), 57 bp (T # 2, sstr2 primer set), and band None (T # 2 sstr2A primer set).

  In FIG. 4, it can be seen that the absence of the antagonist (black line, small square) BN83253 inhibits MCP-1-induced cell migration in a dose-dependent manner. As the concentration of antagonist increases (light gray, large symbol), the BSCI activity of BN832653 is gradually lost.

  In FIG. 5, in the absence of an antagonist (black line, small square), valerolactam inhibits MCP-1-induced cell migration in a dose-dependent manner. As the concentration of antagonist increases (light gray, large symbol), the BSCI activity of BN832653 is gradually lost. Note that this valerolactam BSCI is 30-50 times more potent than BIM83253, so that a higher concentration of competitive antagonist (BIM23627) is required to eliminate BSCI activity from NIM83253 (this graph Is compared with FIG.

  In both FIGS. 6 and 7, BSCI inhibited TNF-alpha production in response to endotoxin in a dose-dependent manner (solid line, square symbols). The apparent efficacy of BSCI was reduced by simultaneous administration of BIM23627 (dotted line, triangle symbol).

  In FIG. 8, dexamethasone inhibited TNF-alpha production in response to endotoxin in a dose-dependent manner (solid line, square symbols). Co-administration of BIM23627 (dotted line, triangle symbol) had no effect on the apparent efficacy of dexamethasone.

  In FIG. 9, two different primer sets were used for both types of cells (see FIG. 3), one producing only amplicons (“A”) from the sstr2a transcript and the other sstr2a and sstr2b ( Here, amplicons of different sizes (“A *” and “B”) are generated from “sstr2”. In FIG. 9, M1 is 398 bp, M2 is 87 bp, and M3 is 57 bp. This data confirms the expression of sstr2a in THP-1 cells and does not provide any evidence for the presence of sstr2b.

  In FIG. 12, the values are measured values (± SD) at three times at each drug concentration. Competition with excess unlabeled BN83250 ("A", 10 nM 3H-BN83250) is shown as a non-specific binding control and the upper dotted line indicating specific binding is determined. A negative control (“B”, excess cold) is provided, which determines the lower dotted line indicating non-specific binding, while comparing the effect of the sstr2 antagonist BIM23627 (“C”; 100 μM) on BN83250 binding. In order to show.

  In FIG. 13, “*” in the polylinker indicates that ATG is upstream of the XbaI site. Note the “ampicillin” and “neomycin” resistant coding reading frames, respectively, used to select for the presence of the plasmid in bacteria and Jurkat cells. The cDNA insert is expressed under the CMV promoter (“P CMV”) in eukaryotic systems and has a standard polyadenylation sequence (“BGH pA”).

  Referring to FIG. 14, (A) shows that BN83250 inhibits SDF-1α-induced migration of Jurkat 3DB (although less potent than that observed in THP-1 cells), but in the parent Jurkat T cell line. It shows that there is little or no effect on SDF-1α-induced migration. For each cell line, the level of cell surface staining of sstr2A measured by flow cytometry is shown in frequency histograms (B) to (D).

  In FIG. 16, compounds that do not affect migration are plotted at a potency of 10 μM and thus appear in the graph. BN83250 (open triangle), (S) -3- (2 ′, 2′-dimethylpropionoylamino) -tetrahydropyridin-2-one (open square), somatostatin (open diamond), and L-779,976 ( White circles) can be identified from the graph. To highlight that all known “BSCI-type” sstr2 agonists are in the lower right and all known “classical” sstr2 agonists are in the upper left, a line of equal identity with ED50 and Ka is shown.

Example 1: sstr2 antagonists block BSCI activity in vitro To demonstrate that known peptides and aminolactam BSCI compounds act through the sstr2 receptor, we used a series of specific sstr2 antagonists. We conclude that if the BSCI action of compounds with different structures is blocked by a series of antagonists for a particular receptor, BSCI activity depends on binding to the receptor blocked by the antagonist used.

  Here we show that the ability of various BSCIs to block migration in response to chemokines (standard activity of BSCI) is inhibited in vitro by several antagonists specific for type 2 somatostatin receptor (sstr2) Prove that.

Method:
In these experiments, the human myelomonocytic cell line THP-1 was used, but similar results were obtained with human peripheral blood monocytes. Leukocyte migration was measured using a 96-well format Transwell® assay system from Neuroprobe (Gaithersburg, MD, USA). In principle, this assay consists of two chambers separated by a porous membrane. Place the chemoattractant in the lower compartment and the cells in the upper compartment. After a time incubation at 37 ° C., when the cells are moved towards the chemoattractant, the number of cells in the lower compartment is proportional to the activity of the chemoattractant (relative to a series of controls).

  Although various assay conditions are acceptable to demonstrate chemokine-induced leukocyte migration (as well as the effects of BSCI or BSCI activity by sstr2 antagonists), specific examples are provided here. This method was basically performed as previously described (Reckless & Grainger (1999) Biochem. J. 340: 803-11).

material
The Transwell® assay system was manufactured by Neuroprobe (Gaithersburg, MD, USA).

  The plates used were a ChemoTx ™ plate (Neuroprobe 101-8) and a 30 μl clear plate (Neuroprobe MP30).

  Gay's Balanced Salt Solution was purchased from Sigma (Sigma G-9779).

  Fatty acid free BSA was purchased from Sigma (Sigma A-8806).

  MTT, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, was purchased from Sigma (Sigma M-5655).

  RPMI 1640 without phenol red was purchased from Sigma (Sigma R-8755).

  The THP-1 cell line (European Cell culture Collection) was used as the white blood cell population.

Test Protocol First, a cell suspension to be placed in the upper compartment was prepared. THP-1 cells were pelleted by centrifugation (770 × g, 4 min) and washed with Gay's balanced salt solution (GBSS + BSA) containing 1 mg / ml BSA. This wash was then repeated and the cells re-pelleted and then resuspended in a small amount of GBSS + BSA for counting using, for example, a standard hemocytometer.

The volume of GBSS + BSA was then adjusted according to the number of cells present so that the final density of the cells was 4.45 × 10 ⁶ cells per ml of GBSS + BSA. This secured 100,000 THP-1 cells for each 25 μl of solution placed in the upper chamber of the plate.

In order to demonstrate the reversal of BSCI activity to MCP-1 induced migration, three lots of cells had to be prepared. A suspension of 4.45 × 10 ⁶ cells / ml THP-1 cells was divided into three bottles. In one bottle, BSCI was added at the appropriate final concentration in an appropriate vehicle (eg, (S) -3- (2 ′, 2′-dimethylpropanoylamino) -tetrahydropyridin-2-one was added to GBSS. From the stock solution in) at 1 nM). In the second bottle, BSCI was added at the same concentration as the first bottle, but also sstr2 antagonist was added (eg BIM23454 [Tulipano et al. (2002) 143: 1218-24] or BIM23627 in stock in GBSS Added from solution at 66 nM). To the third bottle, GBSS + BSA + vehicle was added as appropriate so that the volume in the three bottles acted equally as a control.

  Next, a chemoattractant solution was prepared for placement in the lower compartment. MCP-1 was diluted with GBSS + BSA to a final concentration of 25 ng / ml (after all additions). This is the concentration of MCP-1 that induces maximal migration in this system, as recommended by Frow et al (Med. Res. Rev. 24: 276-98). This solution was divided into three bottles similar to the cell suspension. In one bottle, add BSCI to the same final concentration as added to the cell suspension, and in the second bottle, add both BSCI and sstr2 antagonist to the same concentration as added to the cell suspension. In addition, GBSS + BSA + vehicle was added as appropriate to the remaining bottles to equalize the volume in the three bottles. Note that when establishing the final concentration of MCP-1 in the lower compartment solution and the final concentration of cells in the upper compartment, it is necessary to consider the volume of liquid in order to add the BSCI and sstr2 antagonists I want to be.

  After preparing the chemoattractant solution for the lower well and the cell solution for the upper chamber, the migration chamber was assembled. 29 μl of the appropriate chemoattractant solution was placed in the lower well of the chamber. Each condition was performed by measurement at least three times. After filling all the lower chambers, the porous membrane was applied to the chambers according to the manufacturer's instructions. Finally, 25 μl of the appropriate cell solution was added to each upper chamber. A plastic lid was placed over the entire device to prevent evaporation.

The assembled chamber was incubated for 2 hours at 37 ° C., 5% CO ₂ . Cell suspensions in GBSS + BSA were also incubated in test tubes under the same conditions: these cells build a standard curve to measure the number of cells that migrated to the lower chamber under each condition Used for.

  At the end of the incubation, the liquid cell suspension was gently removed from the upper chamber, 20 μl of ice-cold 20 mM EDTA in PBS was added and the device was incubated at 4 ° C. for 15 minutes. This operation caused all cells attached to the underside of the membrane to fall into the lower chamber.

  After incubation, the filter was carefully flushed with GBSS + BSA to wash away EDTA and then the filter was removed.

  The number of cells that have migrated into the lower chamber under each condition can then be determined by a number of methods, including direct counting, labeling with the use of fluorescent or radioactive markers or vital dyes. Typically we used the vital dye MTT. 3 μl of stock MTT is added to each well, and then the plate is incubated at 37 ° C. for 1-2 hours, during which time intracellular dehydrogenase converts soluble MTT to an insoluble blue formazan product, which is spectrophotometric. Can be quantitatively determined.

  In parallel, an 8-point standard curve was prepared. Starting with the number of cells (100,000) added to each chamber above, serial dilutions were performed 2-fold with GBSS + BSA, 25 μl of cells were added to the plate, and 3 μl of MTT stock solution was added. A standard curve plate was incubated with the migration plate. At the end of this incubation, the liquid was carefully removed from the lower chamber so as not to disturb the precipitated formazan product. After air drying for a short time, 20 μl of DMSO was added to each lower chamber to solubilize the blue dye, and the absorbance at 595 nm was measured using a 96-well plate reader. Next, the absorbance of each well was interpolated into a standard curve to estimate the number of cells in each lower chamber.

  MCP-1 stimulated migration was calculated by subtracting the average number of cells that reached the lower compartment in the well where MCP-1 was not added from the average number of cells that reached the lower compartment where MCP-1 was present at 25 ng / ml. Calculated.

The impact of BSCI was calculated by comparing MCP-1 induced migration that occurred in the presence or absence of various concentrations of BSCI. Typically, inhibition of migration was expressed as the percentage of total MCP-1 induced migration blocked by the presence of BSCI. For most compounds, a dose-response graph was measured by measuring the inhibition of MCP-1-induced migration that occurs at various different compound concentrations (typically 1 nM to 1 μM, or more for less active compounds). Built. The inhibitory activity of each compound was then expressed as the concentration of the compound required to reduce MCP-1 induced migration by 50% (ED ₅₀ concentration). Calculate the effect of sstr2 antagonist by comparing MCP-1 induced migration that occurs in the presence of sstr2 antagonist and BSCI to MCP-1 induced migration that occurs in the presence of BSCI alone (the same concentration of BSCI compound) did. If the sstr2 antagonist inhibited the BSCI activity of the BSCI compound, it was expected that more migration would occur in wells treated with both sstr2 antagonist and BSCI than wells treated with BSCI alone.

  Results: MCP-1 (25 ng / ml) induced substantial migration of THP-1 cells (25,000-40,000 in the presence of MCP-1 than in control wells without MCP-1. Many cells migrate to the lower chamber). This migration was inhibited in a dose-dependent manner by a wide range of BSCI. In FIG. 4, inhibition of MCP-1 responds to increasing concentrations of BSCI BN83253 (N-oleoyl-3-aminocaprolactam; Fox et al. (2005) J. Med. Chem. 48: 867-74). , Indicating that THP-1 cell migration was induced (small square). In FIG. 5, inhibition of MCP-1-induced THP-1 cell migration in response to increasing concentrations of valerolactam BSCI (S) -3- (2 ′, 2′-dimethylpropionylamino) -tetrahydropyridin-2-one (Small square). In both cases, the presence of 600 nM of the sstr2 antagonist BIM23627 (approximately 100 times higher than the Ki of this compound in sstr2; Tulipano et al. (2002) 143: 1218-24) completely prevented BSCI-mediated inhibition (ie Regardless of the concentration of BSCI added, the presence of high doses of BIM23627 restores MCP-1-induced migration to control levels; triangles in FIGS. 4 and 5).

  At low doses of BIM 23627 (eg, 6 nM close to Ki for this compound acting at this receptor; medium size diamond symbol in FIGS. 4 and 5), BSCI action was inhibited by low doses of BSCI. However, at high doses THP-1, inducible cell migration was still inhibited. This is consistent with competition between BSCI and sstr2 antagonists for binding to the same site. At low BSCI concentrations, sstr2 antagonists bind and no inhibition of migration is seen. As the BSCI concentration increases, sstr2 is gradually occupied by BSCI and not by sstr2, and BSCI activity appears.

  At very low doses of BIM 23627 (eg 0.06 nM lower than Ki for this compound acting at sstr2; 0.06 nM; small diamond in FIGS. 4 and 5), BSCI action is seen at all concentrations of BSCI compound However, a low degree of reversal was seen at low concentrations of BSCI, again consistent with competition between these substances binding to the same site.

  These results indicate that different BSCIs ((S) -3- (adamantane-1-carbonylamino) -caprolactam (see WO 2006/016152) and peptide 3 (Reckless & Grainger (1999) Biochem. J. 340: 803-11) Has been reproduced many times. Furthermore, the same results have been obtained using different sstr2 antagonists (BIM 23454; Tulipano et al. (2002) 143: 1218-24). Therefore, for a wide range of BSCI compounds of very different structural classes ranging from peptides to aminocaprolactams and third generation aminovalerolactams, BSCI activity is closely related receptor inhibited by sstr2 (or both antagonists used) ).

  Similar experiments have been performed using antagonists that are selective for other somatostatin receptors. For example, BIM 23627 (provided by Ipsen (Paris, France)), a selective antagonist for sstr3 and sstr5, caused almost a reversal of BSCI activity, even at concentrations 10 times higher than the target receptor Ki. No (<10%).

Example 2: sstr2 antagonists block BSCI activity in vivo In vitro BSCI activity is mediated by sstr2 (or closely related receptors inhibited by BIM23627 and BIM23454). We next tried to demonstrate that the anti-inflammatory activity of BSCI in vivo is also sstr2-dependent. Fortunately, BIM23627 has proven to be active in vivo and can block classical sstr2-mediated signals at least shortly after acute high dose administration (Tulipano et al. (2002) 143: 1218 -twenty four). Therefore, if an acute inflammation model is employed, it could be determined whether the anti-inflammatory effect of BSCI was inhibited by simultaneous sstr2 inhibition.

  We chose this model because the non-lethal model of endotoxemia in mice has been characterized in detail in our laboratory (eg Fox et al. (2002) J. Med. Chem. 45 : 360-70; Fox et al. (2005) J. Med. Chem. 48: 867-74). The extent of systemic inflammation in response to a single intraperitoneal injection of bacterial lipopolysaccharide (LPS) can be estimated by measuring serum TNF-alpha 2 hours after antigen challenge. Furthermore, in this model, BSCI is known to potently inhibit inflammation (Fox et al. (2005) J. Med. Chem. 48: 867-74).

  Methods: Mice (CD1 strain; adult males; 6 mice per group) were treated with BSCI (in the appropriate vehicle) or vehicle alone by the appropriate route. For example, BN83250 (see FIG. 1) is administered by subcutaneous injection (because of poor oral bioavailability), while (S) -3- (2 ′, 2′-dimethylpropionylamino) -tetrahydropyridin-2-one is administered. Forced oral administration. At the same time, one set of mice at each BSCI concentration was administered 4 mg / animal (120 mg / kg) BIM23627 by subcutaneous injection (known to suppress classical sstr2-mediated signaling (eg, GH release) in rats). Treatment conditions; Tulipano et al. (2002) 143: 1218-24). Inflammatory antigen stimulation was induced by intraperitoneal injection of 750 μg LPS (Sigma; L4130 lot # 024K4077; 900,000 EU / mg) 30 minutes to 1 hour after administration (the time when the maximum plasma concentration of BSCI was achieved) It was.

Two more hours later, the animals were sacrificed by CO ₂ asphyxiation and blood was collected by cardiac puncture. Serum was prepared in a conventional manner and the level of TNF-alpha in the serum was measured using an ELISA (R & D Systems; Murine Quantikine code # MTA00) according to the manufacturer's instructions.

  Results: After analysis, the average of animals in each group was reported (data points higher or lower than 3 standard deviations from the remaining points were excluded). As expected, LPS challenge stimulated massive systemic inflammation at levels of TNF-alpha reaching an average of over 3,000 ng / ml (in serum from control animals that did not receive LPS challenge) <Comparison with 10 ng / ml). This inflammation was dramatically inhibited in a dose-dependent manner by treatment with BSCI, regardless of the BSCI used (FIG. 6 shows BN82350 [FIG. 1]; FIG. 7 shows (S) -3- (2 ', 2'-dimethylpropionylamino) -tetrahydropyridin-2-one [valerolactam]).

  Co-administration of the in vivo active sstr2 antagonist, BIM 23627, significantly reduced the anti-inflammatory properties of BSCI at all doses tested (dotted line, triangles in FIGS. 6 and 7). This observation was repeated twice for each BSCI in separate experiments. We expect that co-administration of BIM23627 will reduce the anti-inflammatory potency of BSCI by 10-100 fold (regardless of the BSCI employed). These results clearly demonstrate that in vivo BSCI activity is dependent on sstr2 (or a closely related receptor inhibited by BIM23627), just as we observed in vitro (see Example 1). ing.

  In another series of control experiments, the effect of BIM23627 on the anti-inflammatory activity of dexamethasone, a synthetic corticosteroid that exhibits anti-inflammatory activity by a mechanism completely independent of BSCI, was examined. Dexamethasone reduced serum TNF-alpha levels (as a marker for systemic inflammation) to the same extent as BSCI (compare FIG. 8 with FIGS. 6 and 7), but dexamethasone was slightly more potent at the very high dose (BSCI BSCI was slightly more potent (inhibition of TNF-alpha than 90% compared to about 80% inhibition at the maximum dose), and BSCI was slightly more potent (3-10 times lower than dexamethasone at a dose of TNF-alpha). Statistically significant decline). However, despite the amount of activity in this model, co-administration of BIM23627 had no effect on the anti-inflammatory properties of dexamethasone (dotted line and triangle in FIG. 8). We therefore concluded that BIM23627 has no specific effect on the inflammatory pathway and that BSCI (except for other anti-inflammatory drugs acting by different mechanisms) is highly dependent on sstr2 in vivo.

Example 3: sstr2 expression correlates with BSCI susceptibility The use of multiple sstr2 specific antagonists has been linked to interaction with sstr2 or to different receptors that are inhibited by the same antagonist (closely related to sstr2). It may be dependent on To further demonstrate that sstr2 is a molecular target of BSCI, we measured sstr2 levels by flow cytometry and by qualitative PCR in a series of different types of leukocytes with different sensitivities to BSCI. . The correlation between the presence of sstr2 and sensitivity to BSCI reinforces the conclusion that BSCI activity requires binding to or activation of sstr2.

  Methods: Flow cytometry using antibodies specific for sstr2 was used to estimate the relative level of sstr2 expression on human peripheral blood leukocytes. Whole blood was treated with Erythrolyse (Serotec cat number: BUF04B) according to the manufacturer's instructions. The obtained total leukocyte population was stained with a rabbit polyclonal anti-human sstr2 antibody (Sigma, Cat code S0694; 1 μg / ml) and then labeled with an anti-rabbit secondary antibody (Sigma F9887; 50 μg / ml) labeled with FITC. Staining in parallel with a group of monoclonal antibodies labeled with rPE against various leukocyte CD markers that can be used to unambiguously identify a specific leukocyte subset. After gating the cell population based on the cDNA marker, the average level of sstr2 staining (minus the isotype control) was reported as the mean ± SEM for triplicate measurements. Cultures of THP-1 and Jurkat cells were analyzed using the same protocol except that the cells were not treated with Erythrolyse.

  PCR was performed using the primer set shown in FIG. 3 to distinguish between sstr2a and 2b transcripts. MRNA was prepared from THP-1 and Jurkat cells using the RNAqueous 4PCR kit (Ambion, Cat code # 1914) according to the manufacturer's instructions. The resulting mRNA preparation is then converted to cDNA using reverse transcriptase (RT), which is then used to sstr2 primer, dNTP (Ambion), PCR buffer, and Taq polymerase. The following PCR reaction was constructed with a total volume of 45 μl. The primer sequences are as follows: sstr2 forward primer 5'GCCAAGATGAAGACCATCAC 3 '(SEQ ID NO: 3), and reverse primer 5'GATGAACCCTGTGTACCAAGC 3' (SEQ ID NO: 4). PCR was performed using the following cycle time and conditions using a Peltier PTC-200 thermal cycler according to the manufacturer's instructions: 94 ° C for 2 minutes, 94 ° C for 30 seconds, 55 ° C for 30 seconds. Second, 30 cycles of 1 minute at 72 ° C, and finally 10 minutes at 72 ° C. At the end of amplification, the products were separated on a 2% agarose gel in the presence of ethidium bromide and visualized with a UV transilluminator (Stratagene Eagle Eye II cabinet). Panc-1 cells known to express sstr2 at high levels (Li et al. (2004) J. Surg Res. 119: 130-137) were used as positive controls. A reaction without reverse transcriptase was used as a negative control (confirm that no false positive signal was obtained as a result of genomic DNA contaminating the mRNA preparation). In addition, primers were designed to cross exon boundaries so that genomic amplicons could be easily distinguished from size.

  Results: sstr2 was detected in several leukocyte populations, consistent with previous reports (Hiruma et al. (1990) Immunology 71: 480-5; Tsutsumi et al. (1997) Cell. Immunol. 181: 41-9 ; ten Bokum et al. (1999) J. Endocrinol. 161: 167-75 and the references inside; Elliot et al. (1999) Eur. J. Immunol. 29: 2454-63; Talme et al. (2001) Clin Exp. Immunol. 125: 71-9; Dalm et al. (2003) Am. J. Physiol. Endocrinol. Metab. 285: E344-53; Lichtenauer-Kaligis et al. (2004) Eur. J. Endocrinol. 150 : 565-77). The relative levels of any fluorescent unit are shown in Table 1 below. Interestingly, THP-1 cells (which are highly sensitive to BSCI) show a high level of surface staining for sstr2, while Jurkat T cells (which are almost completely insensitive to BSCI) ) Indicates a staining level that is virtually undetectable.

  Human peripheral blood leukocytes generally show intermediate sstr2 staining between the two cell lines. Granulocytes (mainly neutrophils) and monocytes express high levels of sstr2, and both types of human peripheral blood leukocytes are known to be sensitive to BSCI. Since T lymphocytes (CD3 +) expressed lower but detectable levels and the entire CD3 + population was uniformly stained, this is likely to be similar in both CD4 + (helper) and CD8 + (killer) T cell populations. is there. In contrast, sstr2 was not detected on the B lymphocyte population, a cell insensitive to BSCI (in agreement with the lack of effect of BSCI on antibody production in response to new antigens in vivo).

Table 1. Relative level of sstr2 cell surface staining estimated by flow cytometry on various leukocyte populations. ^a SSC indicates gate control based on side scatter. This is because granulocytes have characteristically high light dispersibility as a result of their cytoplasmic granules.

  The presence of sstr2 mRNA was confirmed in THP-1 cells (FIG. 9) and proved by qualitative PCR to be absent in Jurkat T cells. This confirms the expected specificity of the antibody reagent in these tests. Furthermore, we have demonstrated that the majority of sstr2 present in THP-1 cells is the sstr2a isoform (based on the splicing pattern of mRNA in these cells; FIGS. 3 and 9). Although it is unclear whether the antibodies used in flow cytometry experiments distinguish between sstr2a and sstr2b isoforms, the results shown strongly suggest that the antibodies can detect the sstr2a isoform.

  We concluded that the presence of sstr2 on the leukocyte plasma membrane correlates with susceptibility to BSCI. In particular, it is noted that cells without detectable sstr2 expression are insensitive to BSCI-mediated inhibition of chemokine-induced cell migration.

Example 4: BSCI binds to sstr2 We investigated the molecular interaction between BSCI and the sstr2 receptor using a conventional equilibrium binding assay using labeled BSCI ligands. For these tests we used aminolactam BSCI BN83250, which can be readily synthesized in a form labeled with tritium (ie [ ³ H]) by catalytic hydrogenation of unsaturated precursors (FIG. 10). The unsaturated precursor (S, E) -3- (2 ′, 2′-dimethyldodec-4′-enoylamino) -caprolactam was synthesized as previously reported (WO2005 / 053702).

Method: (S, E) -3- (2 ′, 2′-Dimethyldodec-4′-enoylamino) -caprolactam by catalytic hydrogenation using tritium gas over palladium catalyst (CRB Ltd, Cleveland, UK) [ ³ H] -BN83250 was synthesized and the tritiated product was obtained at a concentration of 270 μM in ethanol with a specific activity of 220 Ci / mmol. (S, E) -3- (2 ′, 2′-dimethyldodec-4′-enoylamino) -caprolactam was prepared as follows: (S, S) -3-aminocaprolactam hydro- in water (30 ml) Pyrrolidine-5-carboxylate (10 mmol) and Na ₂ CO ₃ (30 mmol) are added to a solution of 2,2-dimethyl-dodec-4-enoyl chloride (10 mmol) in dichloromethane (30 ml) at ambient temperature and the reaction is added. Stir for 12 hours. The organic layer was then separated and the aqueous layer was extracted with additional dichloromethane (2 × 25 ml). The combined organic layers were dried over Na ₂ CO ₃ and concentrated under vacuum. The residue was purified by silica column chromatography (1: 1 EtOAc: hexane to EtOAc) to give (S, E) -3- (2 ′, 2′-dimethyl-dec-4′-enoyl) amino-caprolactam colorless. As an oil (2.12 g, 63%). Radiolabeled compounds were stored at 4 ° C. in the dark until needed. Prior to use, specific activity was adjusted by adding unlabeled BN83250.

For analysis of binding, 1 × 10 ⁶ THP-1 cells in Gay's balanced salt solution, 10 mM [ ³ H] -BN83250 (50 Ci / mmol), and various concentrations of (S) -3- ( Individual reactions were constructed using 2 ′, 2′-dimethylpropionoylamino) -tetrahydropyridin-2-one (10 pM to 10 μM) (measured three times for each condition). Excess (100 μM) unlabeled BN83250 was used to measure the level of non-specific binding. After 2 hours at 4 ° C. (conditions already proven to achieve equilibrium binding and this prevents the incorporation of radiolabel that causes overestimation of binding), the excess label is centrifuged (5 ml Gay Washed 3 times with a balanced salt solution of (2) and washed, and the bound radioactivity was measured by scintillation counting of the solubilized cell pellet.

Results: [ ³ H] -BN83250 shows specific and competitive binding to THP-1 cells under equilibrium conditions. Scatchard analysis of binding is consistent with one binding site present at 100,000 to 200,000 sites per cell with an affinity of about 80 nM (FIG. 11). Specific binding of BN83250 to THP-1 cells is inhibited 60-80% (depending on the experiment) by excess (100 μM) of the sstr2 antagonist BN23627 (FIG. 12). Little specific binding was detected in Jurkat T cells lacking sstr2 (<20,000 sites / cell), but specific binding was restored after stable transfection with a vector expressing sstr2 (sstr2 See Example 5 for the preparation of a stable Jurkat T cell line that expresses. Taken together, these results demonstrate that this binding site is sstr2 and is the binding interaction responsible for the observed BSCI activity of BN83250.

(WO 2009/016390) (S) -3- (2 ′, 2′-dimethylpropionoylamino) -tetrahydropyridin-2-one, which we have previously described as one of the most potent BSCIs, The action of competing at the binding site is shown in FIG. (S) -3- (2 ′, 2′-dimethylpropionoylamino) -tetrahydropyridin-2-one competes with [ ³ H] -BN83250 in a dose-dependent manner for binding at sstr2, The ED50 is about 50 pM and the Hill slope is about -1.0, suggesting simple mode competitive binding. High levels of (S) -3- (2 ′, 2′-dimethylpropionoylamino) -tetrahydropyridin-2-one (> 1 nM) inhibit BN83250 binding by 80-90% and are based on two molecules Suggest that they share the same binding site. Peptide 3 (Reckless & Grainger (1999) Biochem. J. 340: 803-11), NR58-3.14.3 (Reckless et al. (2001) Immunology 103: 244-54; WO99 / 12968), (S) -3- (adamantane-1′-carbonylamino) -caprolactam (WO 2006/016152) and (S) -3- (1′-methionine cyclohexyl-1′-carbonylamino) -caprolactam (WO 2006/134384), Other BSCI compounds tested also compete as much as BN83250 for binding to sstr2.

  The apparent affinity constant for binding of (S) -3- (2 ′, 2′-dimethylpropionoylamino) -tetrahydropyridin-2-one to THP-1 cells is determined by in vitro assay (see Example 1). This is consistent with the picomolar ED50 for BSCI activity at 5 but is several orders of magnitude lower than the ED50 for classical sstr2 agonist activity (see Example 7). We concluded that all the groups of BSCI described to date bind to sstr2, and that this interaction is responsible for the BSCI activity of the compounds.

Example 5: Expression of sstr2 confers BSCI sensitivity to cell lines Here we demonstrate that (forced) expression of the sstr2 receptor is necessary and sufficient to confer sensitivity to BSCI. We have already correlated cell surface expression of sstr2 as measured by flow cytometry with sensitivity to BSCI, so cells with little or no sstr2 are insensitive to BSCI but have high levels of sstr2. Cells have already been shown to be sensitive to BSCI-mediated inhibition of chemokine-induced migration. Higher levels of sstr2 are associated with increased potency of BSCI action. However, this correlation has not definitively demonstrated that sstr2 is responsible for BSCI activity.

  We used recombinant DNA technology to force ectopic expression of sstr2 in the Jurkat T cell line, which is a white blood cell line (which normally does not express sstr2). The achieved expression level was measured by flow cytometry. The sensitivity of the stably transfected cell line to BSCI was then measured and compared to the parent Jurkat T cell line.

  Method: Total mRNA from Panc-1 cells (human pancreatic cell line known to express high levels of sstr2) using RNAqueous 4PCR kit (Ambion, Cat code # 1914) according to manufacturer's instructions Was prepared. The mRNA was then reverse transcribed (using RETROscript kit; Ambio) to obtain a cDNA preparation. The PCR (Peltier PTC-200 thermal cycler was then used according to the manufacturer's instructions using the following cycle time and conditions: 94 ° C for 2 minutes, 94 ° C for 30 seconds, 55 ° C for 30 seconds. The full length cDNA of the sstr2 gene was obtained by second, 30 cycles of 1 minute at 72 ° C. and finally 10 minutes at 72 ° C., and was isolated by preparative gel electrophoresis. The sequence of the sstr2 cDNA was confirmed by sequencing. The sstr2 cDNA was treated with EcoRI and XhoI to obtain viscous ends. This allowed directional cloning of the plasmid pcDNA3.1 + (Invitrogen) to the polylinker and subsequent ligation (10 μl ligation was performed using 400 T4 DNA ligase (New England Biolabs) in ligase buffer. The maximum amount of DNA with an insert: plasmid ratio of 3: 1 was incubated overnight at 13 ° C.). The resulting vector (see FIG. 13) was used to transform XL-10 Ultracompetent cells (Stratagene) by standard methods. Plasmid DNA was prepared using the MiniPrep kit (Wizard Plus SV Minipreps DNA purification System; Promega) according to the manufacturer's instructions for transfection into Jurkat T cells.

Jurkat T cells were cultured in RPMI + 10% fetal calf serum (FCS) and subcultured at a dilution of 1: 3 with fresh medium every 4 days. Logarithmic growth phase Jurkat T cells 48 hours after subculture were washed by centrifugation and resuspended in serum-free RPMI medium at 7.5 × 10 ⁵ cells / ml. Next, a mixture of DNA and transfection reagent (2 μg DNA; 3 μl Fugene 6 transfection reagent; Roche) was added and the cells were incubated at 37 ° C. for 48 hours. The cells were then centrifuged 3 times with RPMI + 10% FCS and then washed 3 times, then suspended in fresh medium at 1 × 10 ⁵ cells / ml. Next, 68 ml of the resulting cell suspension was seeded in a total of 226 wells in a 96 well plate (Corning). After 48 hours, G418 (0.9 mg / ml Active G418) was added to the medium to select the transfectants, and subculture was repeated to expand the cells, and the population from each well (clonal or oligoclonal) Were considered separately). After expanding the cell population to a 25 cm ² flask (total of about 5 × 10 ⁶ cells), a small sample was removed and tested for sstr2 expression by flow cytometry.

  Thus, many cells expressing high levels of sstr2 on the cell surface detected by flow cytometry using human sstr2 antibody (Sigma, S0694; see Example 3 for details of the flow cytometry protocol used). Individual cell lines were obtained. One of these strains (named Jurkat-D3B) expresses high levels of sstr2 comparable to peripheral blood monocytes (maximum expression cell population in healthy human peripheral blood; see Example 3). However, the level of sstr2 in Jurkat-D3B was still much lower than in THP-1 cells (FIG. 14).

  Results: The level of CXCR4, the SDF-1α receptor, was the same in the Jurkat-D3B cell line and the parent Jurkat T cell line. In addition, Jurkat-D3B cells responded to SDF-1α over the same concentration range as the parent cell line in the Transwell® chemotaxis assay (see Example 1 for the method used). We concluded that Jurkat T cells and Jurkat-D3B cells showed the same chemokine-induced migration under the conditions of this experiment and are therefore suitable for comparison of their sensitivity to BSCI.

  Jurkat T cells and Jurkat-D3B cells were induced and migrated in response to 100 ng / ml SDF-1α (R & D systems; the concentration of SDF-1α that induced maximum cell migration in both cell lines). BSCI BN83250 was then added to the top and bottom compartments at various concentrations from 0 nM to 1000 nM (each measured in triplicate) (at the same concentration in each compartment so as not to produce a gradient of BSCI concentration). DMSO (1% final concentration) was used as the vehicle and was present at an equal concentration in all wells.

  Consistent with previous results (see Example 3), parental Jurkat T cells expressing low or undetectable levels of sstr2 (FIG. 14) were substantially insensitive to BN83250 (THP− Compared to the ED50 <1 nM for this BSCI on sensitive cells such as one cell line, the ED50 was significantly higher than 1 μM). In contrast, Jurkat-D3B cells expressing moderate levels of sstr2 (FIG. 14) were substantially more sensitive to BN83250 (FIG. 14). The ED50 of BN83250 on Jurkat-D3B was about 10 nM, an increase of more than 100-fold in BSCI sensitivity as a result of ectopic expression of sstr2. Transfection and selection of pcDNA3.1 without insert resulted in a Jurkat T cell line that was insensitive to BN83250 as well as the parent Jurkat T cells.

  Jurkat-D3B also differs in structure, such as peptide NR58-3.14.3 and valerolactam (S) -3- (2 ', 2'-dimethylpropionoylamino) -tetrahydropyridin-2-one Although sensitive to inhibition by other BSCIs, in each case, its potency was significantly lower than on THP-1 cells. This probably reflects the low level of sstr2 expression achieved in Jurkat-D3B compared to THP-1 cells (FIG. 14). It is known that the efficacy of response to pharmacological agents correlates with the level of receptor expression (as a result of so-called “spare receptors”). Our data is consistent with the proposal that sensitivity to BSCI depends on the level of sstr2 expression on the cell surface.

  We concluded that sstr2 expression is necessary and sufficient to confer sensitivity to white blood cells to BSCI.

Example 6: Traditional sstr2 agonists do not have BSCI activity in vitro It has been demonstrated that various structurally different compounds having BSCI activity act through sstr2. (Sstr2 has been identified as a molecular target for the activity of these BSCIs. See Examples 1-5.) All compounds with sstr2 have BSCI activity or have been functionally identified as BSCI. To determine if it is a special class of sstr2 agonists, we tested a series of classic somatostatin receptors for in vitro BSCI activity.

  Method: A leukocyte migration assay was performed exactly as described in Example 1. Briefly, THP-1 cells were induced to migrate in response to 25 ng / ml MCP-1 in a ChemoTx ™ 96 well plate migration system (Neuroprobe Inc., Gaithersburg, MD). Various classical sstr2 agonists were added to different wells (measured in triplicate) at equal concentrations in both the upper and lower compartments (exactly as in the previous experiment demonstrating the acylaminolactam BSCI activity). Reckless & Grainger (1999) Biochem. J. 340: 803-11; Fox et al. (2002) J. Med. Chem. 45: 360-70; Fox et al. (2005) J. Med. Chem. 48: 867-74). After 2 hours at 37 ° C., the number of cells that migrated to the lower compartment was examined exactly as in Example 1 using MTT, a vital staining dye, and standard curve interpolation.

  Results: The natural peptide ligand sstr2 somatostatin (sst14) only reproduced BSCI activity only slightly (FIG. 15). Of note, even at the highest dose of sst14 (1 μM), the maximum inhibition of leukocyte migration achieved was 40-60%, depending on the specific experiment. At sst14 concentrations higher than 10 nM, all sstr2 receptors on the cells are occupied and therefore cannot completely block migration, which is a qualitative difference in how sstr2 signals in response to sst14 compared to BSCI. To express.

  Furthermore, sst14 only inhibits leukocyte migration at concentrations higher than the binding constant of sstr2 binding (approximately 0.05 nM), but all BSCIs tested to date inhibit leukocyte migration at concentrations below their binding affinity. To do. These observations suggest that BSCI induces a stronger signal (with respect to leukocyte migration) than the classical sstr2 agonist (eg, the natural ligand sst14). As a result, BSCI only needs to occupy only a few of the cell surface receptors to maximally inhibit leukocyte migration, whereas conventional sstr2 agonists must occupy essentially all receptors, yet maximal inhibition. Is not achieved.

  Similar experiments were performed using separately processed sstr28 natural ligands with similar results. Leukocyte migration was inhibited only at concentrations higher than the binding affinity at sstr2, with maximal inhibition being only 40-60% even at very high doses. Again, using the synthetic somatostatin analog lanreotide (Prevost et al. (1991) Endcrinology 129: 323-9), which is clinically used to suppress growth hormone release in acromegaly Results were obtained.

  We also tested cortisatin, a recently described somatostatin analog (de Lecea et al. (1996) Nature 381: 242-5), whose receptor binding properties have not been fully determined. Cortisatin may be a different class of natural ligands at sstr2. However, we find that cortisatin is very similar to somatostatin on leukocyte migration (it only inhibits about 50% migration even at very high doses), but much lower It was found to be effective (ED50 = about 75 nM; Table 2). Thus, to date, natural ligands for the sstr2 receptor have not been able to reproduce the strong BSCI activity of acylaminolactams acting at the same receptor.

  Finally, we tested a series of synthetic sstr2 selective and specific agonists provided by Ipsen (Paris, France) and referred to as “BIM” in Tables 2 and 3 below. Somatostatin binds to all somatostatin receptors with broadly similar affinities, and weak BSCI-like activity may be due to opposite signals generated through different receptors. However, with the exception of BIM23065, which inhibited leukocyte migration at 10 μM (but not at lower doses), none of these selective or specific conventional sstr2 agonists were highly active in leukocyte migration inhibition assays. (Table 2).

  Table 2. Various known or suspected sstr2 agonist compounds binding constant (“Ka”) at sstr2 and ED50 for inhibition of MCP-1-induced THP-1 cell migration. Also shown is the maximum inhibition ("maximum effect") observed at the highest dose tested (1-10 [mu] M).

  The affinity of cortisatin with sstr2 has not been clearly reported. BIM23065 inhibits cell migration by 85% at the maximum possible concentration tested (10 μM) and it is therefore unclear whether this is the maximum effect that can be achieved. BIM23A387 is described in Pawlikowsky et al. (2007) J. Physiol. Pharmacol. 58: 179-88 and BIM23120 is described in Ferrante et al. (2006) Endocrine-Related Cancer 13: 955-62.

  Taken together, these results summarize that BSCI is a special class of sstr2 agonists, and most compounds (including natural ligands) inhibit leukocyte migration (and thus in vivo) than the substance known as BSCI. It shows a much lower ability (showing anti-inflammatory activity). The extent to which BSCI differs from classical agonists is shown in FIG. 16, which plots affinity at sstr2 versus ED50 for inhibition of chemokine-induced leukocyte migration. BSCIs gather together and are significantly separated by conventional agonists. Thus, this combination of tests can be used to unambiguously identify new and structurally distinct BSCI groups (no structural similarity to known BSCIs).

  In addition, the method presented here is based on the natural ligand sst14 and its clinical analog lanreotide (Paran & Paran (2003) Curr. Opin. Invetig. Drugs 4: 578-82; Sener et al. (2005). Peptides 26: 493-9; Gonzales-Rey et al. (2007) Ann. Rheum. Dis. 66: 582-8 and the references within; Armani et al. (2007) J. Leukoc. Biol. 81: 845-55 From the weak and less clinically useful anti-inflammatory activity proposed for several somatostatin receptor agonists, including Cury et al. (2008) Dig. Dis. Sci. 53: 2516-20) It is clear to distinguish.

  Existing methods for identifying BSCIs basically consist of functional assays that identify hits, along with traditional medicinal chemistry methods to obtain additional leads around the original hit structure based on defining structure-activity relationships. Dependent on the random screen used. The methods described herein are much more useful because they allow resource-intensive low to medium throughput functional assays to be replaced with high throughput, low cost molecular screening. In this practice of the invention, compounds are first selected based on their interaction with sstr2, and then a relatively small number of positive hits are screened for leukocyte inhibitory activity to identify new BSCIs. This application is further illustrated in Examples 8 and 9 below.

Example 7: BSCI Inhibits Leukocyte Migration More Strongly than Other Conventional sstr2 Targets Although BSCI is an agonist for the sstr2 receptor, not all sstr2 receptor agonists exhibit BSCI activity. Here we examined whether the reverse is true: does BSCI have potent sstr2 agonist-like activity as assessed by other functional assays?

  Two different assays for “classical” sstr2 function were employed: (1) cultured fetal rat pituitary cells secreting growth hormone that was dose-dependently inhibited by sstr2 agonists, and (2) sstr2 A glucose sensitive subclone of the human Panc-1 cell line that secreted insulin that was dose-dependently inhibited by agonists was selected.

Methods: Fetal rat pituitary cell cultures were accurately established by enzymatic dispersion as previously described (Murray et al. (2004) J. Clin. Endocrinol. Metab. 89: 3027-32). After 48 hours in low glucose DMEM + 10% FCS, cells in 48-well plates (5 × 10 ⁴ cells / well) were washed with serum-free DMEM for 4 hours, then in serum-free DMEM for another 24 hours at various concentrations. Incubated with test substance. At the end of this time, conditioned medium was harvested and cell debris was removed by centrifugation and measured for GH using a commercial ELISA (Linco; cat # EZRMGH-45K) according to the manufacturer's instructions.

The glucose-sensitive subclone of human Panc-1 (a cell line derived from polymorphic epithelial carcinoma of the exocrine pancreas) was basically described by Hamil et al. (2008) Biomed. Sci. Instrum. 44: 441-6. Got. Briefly, Panc-1 cells were subcultured in 24-well plates and the medium was supplemented with 5% glucose. After 72 hours, the wells that showed the most significant change to a glucose-sensitive round phenotype were selected and expanded with normal medium. The resulting substrain was then tested for glucose sensitivity by preparing wells (5 × 10 ⁴ cells / well) in a 96-well plate, which was then cultured in culture medium (low glucose) containing a series of glucose concentrations. DMEM, DMEM, DMEM + 1% glucose, DMEM + 2.5% glucose, and DMEM + 5% glucose). After 24 hours, conditioned medium was collected and insulin secretion was measured by ELISA (Linco; cat # EZHI-14K) according to the manufacturer's instructions.

Has the highest glucose sensitivity (3.4-fold increase in insulin secretion in 5% glucose compared to low glucose DMEM) and expresses sstr2 by flow cytometry using the method described in Example 3 Using a proven clone (Panc-2H4), we tested the ability of various sstr2 agonists to regulate glucose-dependent insulin secretion. An additional 96 well plate was prepared using Panc-2H4 (5 × 10 ⁴ cells / well) in DMEM. After 24 hours, the medium was switched to DMEM + 5% glucose containing various concentrations of test substances (3 wells for one condition). After an additional 24 hours, conditioned medium was collected and the insulin concentration was measured by ELISA as described above.

  In both assays, the absence of the effect of the test compound (the highest concentration tested) on the ELISA by including an additional control well in the ELISA exposed to the second highest concentration standard in the presence of each test compound Proved. Results were reported as mean ± SD expressed as fold change from cells in the presence of vehicle alone.

  Results: As expected, a classical sstr2 agonist containing the natural ligand somatostatin-14 reduced GH secretion from rat pituitary cells in a dose-dependent manner with a maximum of 64 ± 9% inhibition at 10 nM. (Table 3). The classical agonist had a similar effect on insulin secretion by Panc-2H4, but the maximal effect was less (43 ± 7% inhibition). In both assays, the concentration required to suppress GH or insulin secretion was consistent with the known affinity of various substances for binding to sstr2.

  In marked contrast, these classical sstr2 agonists do not inhibit migration of THP-1 cells or peripheral blood leukocytes in response to chemokines, or inhibit GH and / or insulin secretion. Inhibition was only at concentrations significantly higher than required (Table 3).

  Peptides 3 and NR58-3.14.3, which are peptides, and aminocaprolactams BN83250 and BN83470, and valerolactam (S) -3- (2 ′, 2′-dimethylpropionoylamino) -tetrahydropyridine-2- Various BSCIs with different structures, including ON, inhibited GH release only at much higher concentrations (Table 3) and had a maximal effect. None of these substances inhibited Panc-2H4 insulin secretion at the maximum concentrations tested. However, as expected, these substances strongly inhibited migration of THP-1 cells and rat peripheral blood leukocytes in response to chemokines (Table 3).

  In summary, the results in Table 3 indicate that compounds that bind to sstr2 and act as agonists at this receptor are easily classified into two groups. One group of substances (referred to as “classical” sstr2 agonists for the purposes of this application) inhibits GH and insulin secretion more potently and potently than chemokine-induced leukocyte migration. A second novel group of substances (referred to as “BSCI type” sstr2 agonists or somatotaxins for purposes of this application) are more potent and potent than chemokine-induced leukocyte migration and inhibit chemokine-induced leukocytes. Inhibits migration.

  Table 3. Comparison of potency of various sstr2 agonists against “classical” versus “BSCI” targets. Shows the potency of action (ED50, in nM) and output (maximum effect as a percent inhibition compared to vehicle) of 12 different sstr2 agonists in 4 separate assays. Two assays measure classical sstr2-dependent responses (GH suppression and insulin suppression), and two assays measure sstr2-mediated BSCI activity (inhibition of leukocyte migration in response to chemokines). The relative potency of these two classes of responses makes it possible to functionally classify sstr2 agonists according to the methods of the invention: the first seven substances are classified as “classical” sstr2 agonists and later The five substances are classified as “BSCI” sstr2 agonists. Abbreviations: SST = somatostatin; n. d. = Not measured; "valerolactam" = (3) -3- (2 ', 2'-dimethylpropionoylamino) -tetrahydropyridin-2-one. BN83470 = (S) -3- (adamantane-1'-carbonylamino) -caprolactam.

  Although no information has yet been disclosed that would have allowed one skilled in the art to recognize that these functionally distinct classes of sstr2 agonists exist, there are several other examples of this phenomenon, here the same receptor Different agonists for can trigger different cellular responses known as “functional selectivity” (for an excellent recent review of this phenomenon at other receptors, see Kenakin (2007) Mol. Pharmacol. 72 : 1393-401). Many molecular mechanisms are the basis for this phenomenon (eg, active state-based selectivity, biased operability, stimulus transfer, etc.), but the nature of the underlying molecular mechanism is described herein. Simple functional assays such as those described in 1) are less relevant to the ability to clearly distinguish group members.

  The group of compounds used here to illustrate the two classes of sstr2 agonists is very clear and easily distinguished (ie, each in terms of functional response generated in several different assays). All members of a class are more similar to each other than members of other classes). However, there may be substances that exhibit “hybrid behavior” across all compound spaces. That is, these substances inhibit GH / insulin secretion and chemokine-induced leukocyte migration with approximately equal potency and output. Such materials would not be easily assignable to any class based on the methods provided herein. We therefore provided a definition that clearly defined the members of each class and excluded “hybrid behavior” compounds from either classification. Has at least 2-fold higher potency (ie, concentration required for half-maximal potency) in GH or insulin secretion assays than chemokine-induced leukocyte migration assay (ie, a ratio greater than 2 in the right column of Table 3) A molecule is defined as a “classical” sstr2 agonist. Conversely, molecules with at least 2 times higher potency or output in the chemokine-induced leukocyte migration assay than the GH or insulin secretion assay (ie, a ratio of less than 0.5 in the right column of Table 3) Defined as “BSCI” sstr2 agonists (somatotaxins) according to the method of the invention.

  Note that in the practice of the methods of the invention, it is preferable to compare assays using cells from the same species. In this example where rat pituitary cells are used to measure the effect of a substance on the classical sstr2 target, the results are compared to the effect of the same substance on chemokine-induced migration of rat peripheral blood leukocytes. Similarly, when human Panc-2H4 cells are used to measure the effect of a substance on the classical sstr2 target, the results are compared to the effect of the same substance on chemokine-induced migration of human THP-1 cells . Applying the method of the invention across species risks the wrong classification of compounds (in both directions) as a result of purely different affinities for sstr2 from the two species. For example, a compound with 10-fold lower affinity in rat sstr2 than human sstr2 was 3 times less potent in the rat pituitary Gh inhibition assay than in the human THP-1 chemokine-induced leukocyte migration assay. In that case, the substance would be misclassified as a “BSCI” sstr2 agonist by the method of the present invention (because correcting for a 10-fold difference in binding affinity for the receptor, the “classical” activity is actually Was higher than “BSCI” activity). For this reason, the ratio of “classical” activity to “BSCI” activity in Table 3 is calculated separately for rats and humans.

Example 8: Yohimbane-16-amide is a functionally selective ligand that acts at the α2-adrenergic receptor Yohimbine is a classic derived from the root of milkweed plants (Rauwolfia Serpentina) It is a member of the class of α-adrenergic mimetics, which also includes reserpine and resinamine. Although these substances are potent antihypertensive drugs (ie, lower blood pressure), they are accompanied by undesirable side effects (which are central to α2-adrenoceptor modulation, which is important for memory and many other neurological functions. Including hypnotic and other mental effects related to action). As a result, yohimbine and reserpine are no longer used as medicines, but are still available as herbal medicines that are widely used as aphrodisiacs in the United States. (This vasodilatory activity causes penile erections as seen in Viagra ™.) However, the risk of hospitalization is high as a result of the antihypertensive crisis, and it is estimated that non-regulated use of herbal extracts including yohimbine causes 10,000 hospitalizations and some deaths each year in the United States) .

  Here we have already described the 16-amide derivative of yohimbine (see WO99 / 12968) which also interacts with the α2-adrenergic receptor, but instead of substantially the classic α2-adrenergic receptor agonist activity. 4 shows that it causes inhibition of chemokine-induced leukocyte migration (ie, BSCI activity). Thus, the 16-amide derivative of yohimbine is a functional selective ligand at the α2-adrenergic receptor (in much the same way that aminolactam BSCI is a functional selective ligand at the sstr2 receptor).

Methods: Radioligand binding experiments were performed according to the manufacturer's instructions using a commercial membrane preparation (Sigma) from cells expressing the α2-adrenergic receptor. [ ³ H] -dihydroergocryptin ([ ³ H] -DHE) at a concentration of 5 nM (0.05 μCi per reaction) radioligand probe (Hoffman et al. (1980) J. Biol. Chem. 255: 4645-52 Used in accordance with the method of The experiment was performed in triplicate and non-specific binding was measured in the presence of excess (10 μM) oxymetazoline.

  Inhibition of MCP-1 induced THP-1 cell migration was measured exactly as described above (Example 1). DMSO (1% final concentration) was used as the vehicle in all experiments.

Results: Yohimban-16-amide inhibited [ ³ H] -DHE binding to the α2-adrenergic receptor in a dose-dependent manner with an apparent Ka = 89 ± 13 nM. This is about 10 times lower than the apparent Ka for binding of yohimbine hydrochloride under the same conditions (6.8 ± 1.2 nM), indicating that yohimbane-16-amide still has α2-adrenergic acceptance with reasonable affinity. Supports binding to the body.

  Similar results have already been obtained using a different α2-adrenergic receptor ligand, RS-79948-197 (Grainger et al. (2005) Mini Rev. Med. Chem. 5: 825-32). The decrease in apparent affinity for the α2-adrenergic receptor due to the introduction of the 16-amide group was greater using this radioligand (770 nM for yohimbane-16-amide compared to 3.8 nM for yohimbine hydrochloride). . This suggests that the yohimbane-16-amide binding site is slightly different from the yohimbine binding site and overlaps the DHE binding site much more than the RS-79948-197 binding site. This small shift of the binding site may be the basis for the functional selectivity we have observed. However, we concluded that yohimban-16-amide binds to the α2-adrenergic receptor.

  Yohimbane-16-amide inhibits MCP-1 induced migration of THP-1 cells (which express significant levels of the α2-adrenergic receptor) with an ED50 of 0.5 nM, whereas yohimbine is Very inactive (ED50 = 30 nM). However, neither substance inhibits SDF-1a-induced migration of Jurkat T cells that do not express α2-adrenergic receptors. Taken together, these results show that both yohimbane-16-amide and, to a lesser extent, yohimbine, exhibit BSCI activity through action on the α2-adrenergic receptors to which they bind.

  Importantly, however, yohimbane-16-amide does not have classical α-adrenergic activity. For example, yohimban-16-amide has no effect on platelet activation in response to ADP, but classical α2-adrenergic ligands (including yohimbine, clonidine, and dihydroergocryptin) are all markedly ADP-mediated Enhances platelet aggregation.

  Taken together, these results clearly demonstrate that yohimban-16-amide is a functionally selective ligand that acts at the α2-adrenergic receptor. Although this compound has less binding to the receptor than yohimbine (because the apparent affinity of competition experiments varies greatly depending on the radioligand probe used, labeled yohimban-16-amide derivatives have been synthesized and characterized. Until then, the actual affinity of the interaction is unknown), but it is still almost 100 times more potent than BSCI. Thus, the binding of yohimban-16-amide promotes the activation of signaling output required for BSCI activity. In contrast, yohimban-16-amide does not enhance ADP-mediated platelet activation (classical α2-adrenergic receptor activity). Consequently, we believe that yohimban-16-amide is an example of a functional selective ligand for GPCR neuroreceptors expressed on leukocytes (BSCI activity substantially replaces classical activity with the receptor). I concluded. Such compounds are potentially very useful as anti-inflammatory agents, without the side effects expected to be associated with target receptor activation, and are readily identified according to the methods of the invention.

Example 9: L-II is a functionally selective ligand that acts at the 5HT2a receptor Amide derivatives of lysergic acid (including N, N-diethylamide, LSD) via agonist activity at the serotonin receptor 5HT2a ( It is a substance that has medically undesired hallucinogenic activity and is therefore banned in many countries as a result of potentially harmful recreational use for its psychoactive effects.

  Here we have also described the lysergic glutamine and asparaginamide conjugates (see WO99 / 12968), which also interact with 5HT2a, but substantially chemokine-induced leukocyte migration instead of classical 5HT2a agonist activity. Inhibition of (ie, BSCI activity). That is, the glutamine and asparagine amide derivatives of lysergic acid are functional selective ligands at the 5HT2a receptor (aminolactam BSCI is the functional selective ligand at the sstr2 receptor, and yohimbane-16-amide is the α2-adrenergic receptor. In much the same way as functionally selective ligands).

Method: Radioligand binding experiments were performed according to the manufacturer's instructions using a commercial membrane preparation (Sigma) from cells expressing the 5HT2a receptor. [ ³ H] -ketanserin as a radioligand probe (according to the method of Leysen et al. (1988) Pharmacol. Exp. Therap. 244: 310-21) at a concentration of 1 nM (0.05 μCi per reaction) (Hoffman et al. al. (1980) J. Biol. Chem. 255: 4645-52). The experiment was performed in triplicate, and nonspecific binding was measured in the presence of excess (10 μM) of metatergoline (a relatively nonspecific ergot-derived serotonin receptor antagonist).

  Inhibition of MCP-1-induced human peripheral blood mononuclear cell migration was measured exactly as described above (Example 1). DMSO (1% final concentration) was used as the vehicle in all experiments.

Results: L-II shows [ ³ H] -ketanserin binding to the 5HT2a receptor receptor in a dose-dependent manner with an apparent Ka = 23 ± 5 nM, similar to the apparent Ka of lysergic acid binding under the same conditions. Inhibited. Thus, L-II binds to the 5HT2a receptor with reasonable affinity, like other amide derivatives of lysergic acid.

  L-II inhibits MCP-1-induced migration of human peripheral blood monocytic cells (which express high levels of the 5HT2a receptor) with an ED50 of 2 nM, whereas lysergic acid is very poor in this assay. Active (ED50> 1 nM). However, neither substance inhibits SDF-1α-induced migration of Jurkat T cells or MCP-1 induced migration of THP-1 cells (both cell lines do not express high levels of 5HT2a). To summarize these results, L-II exhibits BSCI activity through its action on the 5HT2a receptor.

  Importantly, however, L-II has no classical 5HT2a agonist activity. For example, L-II does not induce the traditionally understood neck twist as a hallucinogenic marker in rodents. Most classic agonists of 5HT2a (LSD, mescaline, syrosin, TCB-2, and Bromo-DrangonFLY) induce this classic effect.

  Together, these results clearly demonstrate that L-II is a functionally selective ligand that acts at the 5HT2a receptor. L-II binds to 5HT2a with similar affinity as many classical agonists, but it is still at least 500 times more potent than BSCI. Thus, L-II binding promotes activation of the signaling output required for BSCI activity. In contrast, L-II does not induce neck twist (classical 5HT2a agonist activity). We therefore believe that L-II is an example of a functional selective ligand for GPCR neuroreceptors expressed on leukocytes, where BSCI activity substantially replaces classical activity with the receptor. I concluded. Such compounds are potentially very useful as anti-inflammatory agents, without the side effects expected to be associated with target receptor activation, and are readily identified according to the methods of the invention.

  Functional selectivity at least at the level of intracellular signaling has already been described for the 5HT2a receptor (see, eg, Moya et al. (2007) J. Pharmacol. Exp. Ther. 321: 1054-61), Here, 2,5-DMA induces the release of inositol phosphate via phospholipase C (PLC) activation without activating phospholipase A2 (PLA2), while the associated ligand 2C-N induces PLC Activates release of arachidonic acid by PLA2 without activation. This is consistent with our findings, but there is no teaching of which functional responses differ as a result of this functional selectivity signaling, and there is functional selectivity for a wide range of GPCR neuronal receptors expressed on leukocytes. We do not anticipate our discovery that a series of compounds with useful anti-inflammatory activity can be demonstrated.

Example 10: Identification of candidate BSCIs according to the method of the invention (1)
To further exemplify the method of the invention, it was applied to screen a series of beta-methyltryptophan containing sstr2 agonists related to L-779,976 for BSCI candidates. L-779,976 is one of the early non-peptide sstr2 selective agonists described (Rohrer et al. (1998) Science 282: 737-40), which is a conventional screening method, molecular design. , And a medicinal chemistry approach. This is classically sstr2 agonistic because it is weakly based on the major QFWK tetrapeptide (SEQ ID NO: 5) in the natural somatostatin ligand (see Example 7).

  Interestingly, QxWK (SEQ ID NO: 6) has also been identified as a major motif in the early peptide BSCI (Fox et al. (2002) J. Med. Chem. 45: 360-70), but to date The described non-peptide BSCI sstr2 agonist forms the QxW terminus (see FIG. 1), while the classical sstr2 agonist forms the WK terminus. This leads to the idea that the variant of L-779,976 fused with the glutamine mimetic group is a new class of BSCI.

  Here we tested half a dozen simple new L-779,976 derivatives according to the method of the present invention, one of which (referred to as “Compound C”) is the first member of a new structural class of BSCI compounds. It shows that.

  The six compounds shown in FIG. 17 were transformed into Panc-2H4 insulin inhibition assay (exactly as described in Example 7) at various concentrations from 0.01 nM to 1,000 nM in DMSO vehicle (1% final concentration). And a chemokine-induced THP-1 migration inhibition assay (as exactly described in Example 1). The results are shown in Table 4.

  Table 4. Classical sstr2 agonist and BSCI sstr2 agonist activity of six analogs of L-779,976. Inhibition of glucose-dependent insulin secretion in Panc-2H4 cells was measured exactly as described in Example 7. Inhibition of chemokine-induced THP-1 cell migration was measured exactly as described in Example 1. n. c. = Ratio not calculated (for compounds that show insufficient activity in either assay).

  Parent L-779,976 and Compound A are classical sstr2 agonists. Compounds D, E and F have insufficient activity in either assay to be evaluated according to the method of the invention in this experiment. Compound B has both classical and BSCI sstr2 agonist activity. However, since the inhibitory potency of chemokine-induced leukocyte migration is more than twice as high as that of insulin secretion, the method of the present invention identifies Compound C as a novel and unique structure BSCI compound (somatotaxin).

  Here we show how the method of the invention can be applied to a series of analogs of known sstr2 agonist compounds to unambiguously identify compounds with BSCI activity that are suitable for further development as anti-inflammatory agents. This is an example. Since the knowledge of sstr2 binding was used in the first step to select a small number of molecules to be screened using a functional assay, no experimental screening for binding to sstr2 was performed. It is clear that this is an application of the method.

Example 11: Identification of candidate BSCIs according to the method of the invention (2)
In the previous example, knowledge of the molecular target of BSCI was used to design candidate BSCI compounds for functional screening based on modifications of known sstr2 receptor agonists. However, the method of the invention can also be applied to random screening of very large libraries. In this application, conventional molecular screening using sstr2 as a target is first used to identify hits from a random library, and then in a second stage, the hit (or its analog) is “classic” It is screened using a pair of functional assays for “typical” sstr2 agonist activity and “BSCI” sstr2 agonist (somatotaxin) activity.

Methods: Membranes prepared from Jurkat T cells stably transfected to express sstr2 (Jurkat D3B; see Example 5) were used to establish molecular screening for compounds that interact with sstr2. Briefly, Jurkat D3B cells (5 × 10 ⁷ cells per preparation) were washed by centrifugation in Dulbecco PBS, then 1 ml ice-cold Dulbecco containing protease inhibitors (1 mM PMSF, 100 mM leupeptin). Resuspended in PBS. The cell suspension was then lysed using a Dounce homogenizer and the resulting homogenate was centrifuged at 700 × g for 1 minute at 4 ° C. to pellet unbroken cells and large cell fragments. The supernatant was taken out, transferred to an ultracentrifuge tube, and centrifuged at 60,000 × g for 2 hours at 4 ° C. to obtain a pellet of membrane fragments. The pellet was gently washed with ice-cold Dulbecco-PBS containing protease inhibitors and then resuspended in 100 μl Dulbecco-PBS containing protease inhibitors. The protein concentration was then measured using the Coomassie Plus kit (Pierce) according to the manufacturer's instructions and the final concentration was adjusted to 2 mg / ml by further addition of Dulbecco-PBS containing protease inhibitors. . An equal volume of glycerol was then added as a cryoprotectant (giving a stock preparation of membranes with a total protein concentration of 1 mg / ml in 50% glycerol) and the stock was stored at -80 ° C for up to 1 month until use.

Screening was done in 384 well microtiter plates. In each well, 1 μg Jurkat D3B membrane fraction protein in binding buffer (Gay's balanced salt solution), 1 nM [ ¹²⁵ I] -labeled somatostatin-14 (400 mCi / mmol), and final from 100 mg / ml in DMSO One reaction consisting of test compounds from a 1 mg / ml concentration library was composed of a final reaction volume of 25 μl. Allow to bind for 2 hours at 37 ° C. (conditions already established to reach equilibrium binding), then filter the reaction through a GFC filter sheet in a 384 well manifold, and add 0.5% Triton X-100 and 300 mM NaCl. An additional 10 ml of binding buffer was used to wash in continuous flow. The bound count was assessed by quantitative scintigraphy. In each plate, BIM 23627 (10 μM) was used as a positive control for binding and 100 nM unlabeled somatostatin was used to measure non-specific binding. Binding was expressed as percent inhibition of specific counts in the presence of a compound. When all 384 well plates were assayed and evaluated without the addition of test compound, the suppression of specific counts to 38% was 4 standard deviations below the negative control (vehicle only) level. As a result, a test substance giving a suppression of 76% or more of the specific count was regarded as a positive hit.

  Hits from this assay were confirmed using conventional Scatchard analysis exactly as described in Example 4. If the apparent Ka for binding to sstr2 was 1 μM or more, the compound was further investigated.

  Next, compounds confirmed to interact with sstr2 were evaluated in a paired functional assay: panc-2H4 insulin inhibition assay (estimated as “classical” sstr2 agonist activity performed exactly as described in Example 7). And chemokine-induced THP-1 cell migration inhibition assay (performed as described in Example 1 exactly as an estimate of “BSCI” sstr2 agonist activity). In accordance with the method of the present invention, a substance having at least a 2-fold higher potency in a chemokine-induced THP-1 cell migration inhibition assay is selected as a candidate BSCI suitable for further research and development as an anti-inflammatory agent.

  Results: 26 assay plates were run (9,880 compounds were screened in total), yielding 28 hits (0.28%) that suppressed labeled somatostatin-14 and sstr2 binding by more than 76%. It was.

  Of these primary hits, 11 (39% of the primary hits; 0.11% of the library) were successfully reproduced by Scatchard analysis and 9 (81% of the repeated hits; 0.09% of the library) Had an apparent binding affinity for sstr2 of greater than 1 μM.

  Next, the reproduced hit (referred to as ASX.A-ASX.I in terms of apparent binding affinity for sstr2 and ASX.A has the highest apparent affinity) is exactly as in Example 7. The paired functional assay was evaluated as described and the results are shown in Table 5.

  Table 5.9 Reproduced moderate affinity (1 μM or higher) hit classical sstr2 agonist and BSCI sstr2 agonist activity from screening of compounds in 880. Inhibition of glucose-dependent insulin secretion in Panc-2H4 cells was measured exactly as described in Example 7. Inhibition of chemokine-induced THP-1 cell migration was measured exactly as described in Example 1. n. c. = Ratio not calculated (for compounds that show insufficient activity in either assay). The data for L-779,976 and BN83250 are presented as an example of a classic type sstr2 agonist and a BSCI type (somatotaxin) sstr2 agonist for comparison.

  Surprisingly, 9 out of 11 compounds had detectable agonist activity in either functional assay. For most GPCRs, less than 10% (1 of 10) of the hits identified by high-throughput screening can be expected to have agonist activity. This unusual finding reflects the relative complexity of agonist: receptor interactions with sstr2 or may be the result of a specific screen that we have chosen to establish (compete for binding of natural ligands). I don't know. In addition, the screened library was not random and was already enriched using a template with structural similarity to a known GPCR agonist.

  Three of these nine have high agonist activity that can be clearly identified as having a “classical” type of sstr2 agonist activity according to the methods of the invention (ASX.A, ASX.C, and ASX.D). ), At least twice as potent in the insulin inhibition assay as in the cell migration inhibition assay. In contrast, only one (ASX.H) that had the lowest apparent binding affinity of the hits reproduced (ASX.H) had at least two times higher potency in the cell migration inhibition assay than the insulin inhibition assay. , Specifically identified as a “BSCI” type sstr2 agonist (one sototaxin). These findings indicate that a single compound (ASX.D) was tested for ASX. Has a potency comparable to H, but also has a high potency in the insulin suppression assay for “classical” sstr2 agonist activity, so even a functional assay for inhibition of cell migration is a “BSCI” type sstr2 agonist (somatotaxins) It is exemplified that it cannot be used alone to clearly identify The methods of the present invention are both necessary and sufficient to identify completely new “BSCI” type sstr2 agonists (somatotaxins).

  Using the method of the present invention, we screened 9,880 compounds and were of sufficient nature as BSCI (of course, with an ED50 of 45 nM, it is about 1 in vitro than the optimized lead candidates we have already described. One compound was found that would be able to serve as a template for a traditional medicinal chemistry lead optimization program with a 000-fold lower potency; see eg WO2009 / 016390). Direct screening of all 9,880 compounds by functional assay would have taken months (about 100 compounds can be screened in normal week work). The application of the method of the present invention utilizing knowledge of the molecular target of BSCI made it possible to complete this whole model screening in just one week. This is a reduction of about 99% of the resources consumed. Typically, high-throughput screening performed in the pharmaceutical industry would be able to screen 10 to 100 times more compounds than those we used in this example. We assume that such screening is easy using the method of the present invention, but is not feasible using a functional screening assay and will require almost 10 years to achieve the same screening amount. .

Example 12 In Vivo Evaluation of Candidate BSCIs Identified by the Methods of the Invention To identify agents useful as agents that have anti-inflammatory properties but do not have the endocrine modulating properties of classical somatostatin receptor agonists Compound P was identified according to the method of

  Compound P binds to sstr2 where it acts as an agonist. As shown in FIG. 18, Compound P inhibits THP-1 cell migration in response to the chemokine MCP-1 in an assay according to the method of the present invention with an ED50 of about 0.05 nM.

  However, as shown in FIG. 19, Compound P only inhibited growth hormone (GH) release from cultured pituitary cells at an ED50 of about 2000 nM in an assay performed according to the method of the present invention. In FIG. 19, the dotted line shows the maximal effect (about 75% reduction in growth hormone secretion) achieved with this assay, for example using 10 nM somatostatin-14 (a classical sstr2 agonist). Consequently, the ED50 dose is the dose of Compound P that reduced GH secretion by about 37%.

  Thus, the ratio of classical type activity to BSCI type activity of compound P is calculated to be about 0.000025.

  To demonstrate that assays performed in vitro are predictive of compound activity in vivo, 36 normal healthy human subjects (male or female) were assigned to 3 groups of 12 subjects. Entered clinical trial. Of the 12 subjects in each group, 9 were orally administered Compound P as a liquid drug in 60 mL of water, and the remaining 3 received only 60 mL of water as a placebo. Group 1 was administered 150 mg of Compound P daily for 7 days, Group 2 was administered 500 mg of Compound P daily for 7 days, and Group 3 was administered 1500 mg of Compound P daily for 10 days. Blood samples were collected before, during, and after administration of Compound P.

  Compound P was found to have almost 100% oral bioavailability in humans with a half-life of about 25 hours. The predicted exposure of the subject to Compound P was confirmed using an LC-MS assay evaluated for Compound P in the subject's serum.

  As expected, Compound P was anti-inflammatory in humans. C-reactive protein (CRP) is a well-recognized marker of human inflammatory levels and is elevated during acute and chronic inflammatory responses (see Coventry et al. (2009) J. Translational Med. 7: 102). ). As a result, serum CRP levels are widely accepted as an alternative marker for inflammation. CRP levels were measured using a commercial ELISA assay on serum samples before administration (prior to administration) and the last day of administration (after administration). Treatment with all three doses of Compound P decreased CRP levels in treated subjects, but not in subjects who received placebo. The effect of Compound P treatment (regardless of dose) is shown in FIG.

  FIG. 20 compares the action of Compound P with Medrol® (methylprednisolone, corticosteroid), a well-established clinical anti-inflammatory drug. Both compounds reduced CRP to the same extent, but the effect was statistically significant only for compound P (p = 0.02).

  The results in FIG. 20 confirm that Compound P is an effective anti-inflammatory agent at least as well as the synthetic corticosteroid Medrol®.

  However, as also predicted according to the method of the present invention, we bind compound P to the type 2 somatostatin receptor but have no effect on a panel of markers of the hypothalamic-pituitary adrenal axis (HPA axis) We have therefore found that the classical type of activity is completely lacking in humans.

  Compound P had no effect on GH levels in humans treated for 7 days, even at very high doses. Furthermore, Compound P had no effect on ACTH levels in humans treated for 7 days, even at very high doses. ACTH has been established to be the most sensitive marker of human HPA axis disruption (see Denef (2008) J. Neuroendocrinol. 20: 1-70). Has a clinically useful anti-inflammatory effect in humans, but does not have a classic type of somatostatin mimicking effect at the same dose.

  This example shows that a compound identified according to the method of the invention that binds to the type 2 somatostatin receptor has anti-inflammatory activity that is clinically useful in humans and is (in this connection) an undesirable endocrine modulating effect. (Or “classical type” action).

Summary of Examples To summarize the examples, we have demonstrated that four different groups of BSCIs act as agonists at three different GPCR neuroreceptors expressed on leukocytes. Peptide 3-related polyamides and aminolactams act as agonists of the type 2 somatostatin receptor (sstr2; Examples 1-5). The 16-amide derivative of yohimbine acts via the α2-adrenergic receptor (Example 8). The glutamine and asparaginamide conjugate of lysergic acid acts as an agonist of the 5HT2a serotonin receptor (Example 9). However, in each case, the majority of other agonists for these receptors cannot act as BSCI (Examples 6, 8, and 9) or act with substantially lower potency and / or power. As a result, we concluded that BSCI is a more general class of special, enabled defined subclasses of agonists for these receptors.

  Furthermore, it is reasonable to conclude that such functionally selective ligands exist for all GPCR neuroreceptors since these groups were identified only by their mutual structural relationship. It is surprising that we have discovered functional selective ligands at three different GPCR neuroreceptors expressed on leukocytes (the method used to identify these compounds is Because it did not depend on any information). Since the mechanism of action of these substances has been disclosed (here), this information can be used to find ligands that act similarly at other GPCR neuroreceptors expressed on leukocytes. A method for performing such a search is one embodiment of the present invention.

  It is useful to subclassify agonists with GPCR neuroreceptors expressed on leukocytes based on the functional selectivity of BSCI activity over classical agonist activity (this is independent of the structural relationship to known classes). To allow identification of new BSCIs), new (since not described so far), well defined (illustrated in Examples 10 and 11, clearly distinguishing subclass members) The method is shown) and surprising (all the existing scientific or patent literature is functional of GPCR neuroreceptors expressed on leukocytes that promote anti-inflammatory activity versus classical activity) Does not suggest that there may be clear subclasses).

  In both Example 10 and Example 11, prior information on the molecular targets of BSCI, the subject of the present invention, was used to identify new and unique structure BSCI compounds. In Example 10, targeting information is used to select L-779,976 as an appropriate scaffold for analog generation and identify the most promising candidates for subsequent development as anti-inflammatory agents In order to do so, these analogs were subsequently screened using a pair of assays according to the method of the invention (an assay of “classical” sstr2 agonist activity and an assay of “BSCI” sstr2 agonist activity). In Example 11, target information was used to plan and develop direct molecular screening for sstr2 interactive structures. This was then further refined by applying one assay (an assay of “classical” sstr2 agonist activity and an assay of “BSCI” sstr2 agonist activity) according to the method of the present invention. This identified the most promising library elements for further development as anti-inflammatory agents.

  Finally, in Example 12, a compound identified in the present invention that binds to the type 2 somatostatin receptor has an anti-endocrine regulatory (or “classical type”) action that is clinically useful in humans. It was confirmed that the method of the present invention is effective by demonstrating that it has inflammatory activity.

  The surprising finding that BSCI acts with functional selectivity at GPCR neuroreceptors expressed on leukocytes is a substance with promising BSCI activity as a novel anti-inflammatory agent for treating a very wide range of diseases Provides a unique and useful new method for identifying To date, the only way to identify new and unique structure BSCI compounds is to further modify the structure of existing BSCI compounds (effectively expanding the number of known BSCI compounds within existing structure classes Or screening a library of compounds not selected in the functional assay for BSCI activity (eg, the Transwell® cell migration assay widely used in the examples of the present invention). Compared to tens of thousands of compounds processed by conventional molecular screening in the same period, even the best method available to date can screen only a few hundred new compounds by functional assays. A difference of 2-3 orders of processing speed between molecular and functional screening is important for the usefulness of the present invention: in finding a new structural class of BSCI by random functional screening, the resource problem is impractical It ’s so big. However, the method of the present invention first performs a conventional molecular screening using GPCR neuroreceptors expressed on leukocytes, and then in a second step further screens hits from the molecular screening by functional assays. , To identify the functional properties of BSCI (ie, substances that are more potent in inhibiting chemokine-induced leukocyte migration than in inhibiting GH or insulin release). The first step of molecular screening reduces the number of compounds in the random library by an order of 2-3 (since a reasonable potency hit is typically only 0.1-1% of the random library) ) Subsequent functional screening resource requirements become realistic, although greater utilization per compound to be screened.

  Although the invention has been described with reference to preferred embodiments, various modifications and variations can be made without departing from the spirit and scope of the invention, and such modifications are obvious to the invention. Can be recognized by those skilled in the art. The embodiments disclosed herein and set forth in the claims are by no means limiting and should not be limiting.

  All documents mentioned herein are hereby incorporated by reference in their entirety.

  The sequences listed in the sequence listing below form part of the present invention.

Free text synthesis oligonucleotides of the sequence listing (SEQ ID NO: 3-4);
The major BSCI motif of the natural somatostatin ligand (SEQ ID NO: 5);
The main motif of the early peptide BSCI (SEQ ID NO: 6);
Xaa can be any naturally occurring amino acid (SEQ ID NO: 6).

Claims (29)

A method for identifying a compound or agent having broad spectrum chemokine inhibitor (BSCI) activity, comprising the following steps:
(A) First, one or more candidate compounds or substances are screened for binding to GPCR neuroreceptors known to be expressed on leukocytes;
(B) a compound or substance that exhibits binding to the selected GPCR neuroreceptor is tested for BSCI activity in a functional assay; and
(C) A compound or substance that exhibits binding to the selected GPCR neuroreceptor is tested for classical agonist activity in a functional assay;
Wherein step (a) is performed first, but steps (b) and (c) are performed in any order or simultaneously.   2. The method of claim 1, wherein the functional assay for BSCI activity is an assay for inhibition of chemokine-induced cell migration in vitro.   2. The method of claim 1, wherein the functional assay for BSCI activity is an assay for inhibition of chemokine-induced cell migration in vivo.   2. The method of claim 1, wherein the functional assay for BSCI activity is an assay for cytokine secretion of T helper cells.   5. The method of any one of claims 1-4, wherein the functional assay for classical agonist activity is performed in vitro.   5. The method of any one of claims 1-4, wherein the functional assay for classical agonist activity is performed in vivo.   7. A method according to any one of claims 1 to 6, wherein an alternative biomarker is used to detect the result of BSCI activity.   8. The method of any one of claims 1-7, wherein an alternative biomarker is used to detect the result of classical agonist activity.   9. The method according to any one of claims 1 to 8, wherein the candidate compounds or substances to be screened are selected from or combined with the library.   The method according to any one of claims 1 to 9, wherein the candidate compound or substance to be screened is produced by combinatorial chemistry.   11. A method according to any one of claims 1 to 10, wherein the candidate compound or substance to be tested is a mixture of compounds.   The molecular interaction with the GPCR neuroreceptor is performed using whole cells expressing the selected GPCR neuroreceptor or a membrane derived from a cell expressing the selected GPCR neuroreceptor. Item 12. The method according to any one of Items 1 to 11.   13. The method of claim 12, wherein the cell has been genetically engineered to overexpress the selected GPCR neuroreceptor.   14. A method according to claim 12 or 13, wherein the cell expresses or has been genetically engineered to express a selected fragment of the GPCR neuroreceptor.   15. A method according to any one of claims 1-14, wherein molecular interaction with the selected GPCR neuroreceptor is performed using an isolated or purified receptor.   16. The method according to claim 15, wherein any fragment of the somatostatin receptor isolated or purified is used.   The interaction between a candidate compound or substance and the selected GPCR neuroreceptor is measured by competition with another labeled ligand capable of binding to the selected GPCR neuroreceptor. The method of any one of -16.   The method according to any one of claims 1 to 17, wherein the interaction between the candidate compound or the substance is directly measured.   The method according to any one of claims 1 to 18, wherein the GPCR neuroreceptor is a somatostatin receptor.   20. The method of claim 19, wherein the GPCR neuroreceptor is an sstr2 receptor.   20. The method of claim 19, wherein the somatostatin receptor is sstr2A.   20. The method of claim 19, wherein the somatostatin receptor is sstr2B.   23. A method according to any one of claims 19 to 22, wherein the candidate compound or substance excludes somatostatin.   The method according to any one of claims 1 to 18, wherein the GPCR neuroreceptor is an α2-adrenergic receptor.   25. The method of claim 24, wherein the [alpha] 2-adrenergic receptor is ADRA2A, ADRA2B, or ADRA2C.   The method according to any one of claims 1 to 18, wherein the GPCR neuroreceptor is a serotonin receptor.   27. The method of claim 26, wherein the serotonin receptor is 5HT2a.   28. Any of claims 1-27, wherein an interaction between one or more of said test substances and said GPCR neuroreceptor is measured or inferred from any kind of scientific literature or investigation of existing databases The method according to claim 1.   29. Any one of claims 1-28 comprising an additional step for defining a new structural class of BSCI based on one or more structural motifs present in a compound selected by application of a previous step. The method described in 1.

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